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Hamilton's  System  of 

LEGAL  MEDICINE 


[Complete  in  2  Vols. 

A  Complete  work  of 
Reference  for  Medical 
and  Legal  Practitioners 


By  ALLAN  flcLANE  HAMILTON,  M.D., 

Consulting  Physician  to  the  Insane  Asylums  of  New  York  City, 

Assisted  by  LAWRENCE  QODKIN,  Esq.,  of  the 
New  York  Bar,  and  Others. 


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AN    ATLAS 


OF   THE 


Bacteria  Pathogenic  in  Man 

WITH 

DESCRIPTIONS   OF   THEIR   MORPHOLOGY   AND 
MODES  OF  MICROSCOPIC  EXAMINATION. 

BY 

SAMUEL  G.   SHATTOCK,  F.E.C.S., 

Joint  Lecturer  on  Pathology  and  Bacteriology,  St.  Thomas'  Medical  School, 

London ;  Pathological  Curator  of  the  Museum  of  the 

Royal  College  of  Surgeons,  London. 


WITH   AN  INTRODUCTORY   CHAPTER 
ON 

BACTERIOLOGY  : 

ITS  PRACTICAL  VALUE  TO  THE  GENERAL  PRACTITIONER. 

BY 

W.   WAYNE  BABCOCK,   M.D,, 

Pathologist  to  the  Kensington  Hospital  for  Women;  Clinical  Pathologist  to  the 

Medical-Chirurgical  Hospital;  Demonstrator  of  Pathology  and  Bacteriology 

in  the  Medico-Chirurgical  College  of  Philadelphia. 


SIXTEEN  FULL-PAGE   COLORED  PLATES. 


E.   B.   TEEAT   &   CO., 

241-243  West  23d  Street, 

NEW  YORK, 

1899.  Price,  81. OO 


COPYRIGHT, 
E.  B.  TREAT  &  CO. 

1899. 


J. 


Publisher's  Note. 

This  Atlas  was  originally  published  in  the  "International 
Medical  Annual "  in  two  sections,  one  in  the  1898  issue,  the  other 
in  the  1899  issue. 

The  perfection  of  the  plates  illustrating  the  appearance,  under 
the  microscope,  of  the  Bacteria  Pathogenic  in  the  human  subject 
was  of  such  a  high  degree  as  to  evoke  the  admiration  of  those 
familiar  with  Bacteriology.  Then,  too,  the  descriptive  text  was 
succinct  and  practical,  and  written  for  the  use  of  physicians  desir- 
ous of  keeping  up  to  date,  but  who  have  not  the  time  nor  inclina- 
tion to  master  the  elaborate  text  books  on  Bacteriology. 

We  have  had  requests  that  these  articles  be  reprinted  in  book 
form,  thus  giving  a  sufficiently  complete  work  on  the  subject  to 
meet  ordinary  requirements.  Having  secured  a  number  of  sets  of 
the  illustrative  plates,  we  have  decided  to  meet  these  requests.  We 
trust  our  effort  to  give  the  medical  profession  a  small  but  first-class 
work — both  as  to  authorship,  illustrations,  press  work,  etc. — at  a 
very  reasonable  price  will  meet  with  a  reception  such  as  we  believe 
it  to  be  entitled  to. 

The  value  of  the  original  work  has  been  much  added  to  by  the 
publication  of  an  introductory  chapter  on  "Bacteriology;  its  Prac- 
tical Value  to  the  General  Practitioner,"  by  W.  Wayne  Babcock, 
M.D.,  of  Philadelphia. 


M3517S5 


BACTERIOLOGY : 
Its  Practical  Value  to  the  General  Practitioner. 


Bacteriology :    Its  Practical   Value  to   the 
General  Practitioner. 

BY  W.  WAYNE  BABCOCK,  M.D., 

Pathologist  to   the   Kensington   Hospital  for  Women;    Clinical  Pathologist  to  the 
Medico-Cbirurg'cal  Hospital;  Demonstrator  of  Pathology  and  Bacteriology 
in  the  Medico- Chirurgical  College,  of  Philadelphia. 


To  the  general  practitioner,  bacteriology  is  offering  a  con- 
stantly increasing  field  of  usefulness.  From  its  earliest  days,  this 
science  has  suggested  a  theoretical  basis  for  treatment,  while  it  has 
developed,  especially  during  more  recent  years,  numerous  prod- 
ucts of  practical  remedial  value.  For  the  most  part  it  has  not 
been  difficult  for  the  physician  to  avail  himself  of  these  advances 
in  treatment.  The  methods  founded  upon  theory  have  been  par- 
ticularly popular,  and  it  has  been  only  necessary  for  the  micro- 
organismal  nature  of  an  affection  to  gain  credence  in  order  to 
have  innumerable  preparations  of  real  or  fancied  antiseptic  value 
pressed  into  service. 

From  the  earlier  vague  and  theoretic  means  of  combating 
bacterial  invasion,  we  are  now  emerging  upon  a  more  rational 
therapeusis,  founded  upon  laboratory  investigation  and  endorsed 
by  clinical  trial.  Although  preventive  inoculations  and  the  use  of 
toxins  and  antitoxins  have  been  adopted  with  some  reluctance, 
their  application  is  not  difficult  and  their  employment  is  now  be- 
coming general.  Coincident  with  the  recent  advances  in  etiology, 
prophylaxis  and  therapeutics,  and  scarcely  less  important,  are  the 
strides  made  in  bacteriologic  diagnosis  and  prognosis.  And  yet, 
despite  the  great  value  of  these  latter  innovations,  it  is  entirely 

7 


g  BACTERIOLOGY  : 

probable  that  bacteriology  has  been  of  far  greater  service  to  the 
practitioner  in  the  line  of  treatment  than  in  diagnosis.  The 
scratch  of  the  vaccine  lancet  or  the  thrust  of  the  antitoxin  needle 
requires  neither  erudition  nor  great  technical  skill,  while  in- 
dulgent manufacturers  beg  to  supply  the  practitioner  with  more 
convenient  and  refined  products.  On  the  other  hand,  unfortun- 
ately, the  methods  of  diagnosis  have  not  only  required  laboratory 
training  but  also  laboratory  apparatus. 

These  difficulties  are  being  largely  overcome  in  the  medical 
centers  by  the  establishment  of  municipal  laboratories,  but  in  the 
more  remote  districts  the  general  practitioner  is  left,  as  usual, 
self-dependent.  There  is,  moreover,  a  prevalent  impression  that 
the  general  practitioner  requires  but  little  knowledge  of  bacteri- 
ology. This  needs  correction.  The  specialist  is  a  man  who,  by 
exceptional  proficiency  in  a  single  branch,  is  exempted  from  a 
thorough  knowledge  of  general  medicine.  Serving  in  a  single 
field,  he  exacts  service  from  his  confreres  in  all  others,  including 
that  of  skilled  laboratory  workers.  The  true  position  of  the 
general  practitioner  is,  naturally,  quite  the  reverse,  and  with  the 
knowledge  that  "diagnosis  is  treatment,"  bacteriology  must  be  far 
from  the  least  of  'his  many  accomplishments.  He  may  therefore 
rejoice  in  the  fact  that  some  of  the  most  important  of  bacteri- 
ologic  methods  now  require  but  simple  apparatus,  are  capable  of 
rapid  performance  and  demand  no  exceptional  skill.  Given  a 
good  microscope,  which  may  now  be  considered  an  essential  part 
of  the  practitioner's  outfit,  a  very  moderate  additional  expense 
will  provide  the  essential  equipment  for  many  of  the  very  impor- 
tant diagnostic  tests.  Indeed,  it  is  now  possible  without  any  aid 


ITS    PKACTICAL   VALUE   TO   THE   GENERAL   PRACTITIONER.      9 

from  the  microscope  and  with  no  more  complicated  apparatus 
than  a  test  tube,  to  determine  the  presence  and  activities  of 
typhoid  bacilli  in  the  body;  and  thus,  by  a  procedure  scarcely 
more  difficult  than  the  test  for  sugar  in  the  urine,  diagnose  the 
existence  of  enteric  fever.  The  single  example  may  serve  to  il- 
lustrate the  error  of  a  prevalent  opinion  that  bacteriology  is 
hardly  accessible  to  the  practitioner  as  an  aid  in  his  daily  work. 

In  this  article  I  desire  to  mention  a  number  of  the  practical 
advances  in  bacteriology  and  to  indicate  how  much  of  essential 
value  in  the  various  fields  they  offer  to  the  progressive  physician. 
Certain  of  these  advances  may  never  have  a  general  use;  others 
are  as  yet  very  imperfectly  developed ;  improvements  may  be  ex- 
pected upon  all ;  yet  we  do  our  patients  an  injustice  if  we  do  not 
avail  ourselves  of  some  of  the  present  benefits.  It  is  convenient 
to  group  the  bacteriologic  advances  under  the  general  headings  of 
Etiology,  Diagnosis,  Prognosis  and  Treatment.  In  attempting 
this,  no  excuse  is  offered  for  mentioning  much  that  is  trite  to 
many  medical  readers. 

Etiology. — On  referring  to  the  causal  relations  of  bacteria  to 
morbid  conditions,  we  find  that  there  is  a  large  number  of  dis- 
eases for  which  micro-organisms  may  reasonably  be  claimed  as 
the  inducing  factors;  a  considerable  number  for  which  certain 
bacteria  have  been  described  as  the  etiologic  agents,  and — until 
within  a  very  recent  period — but  a  very  moderate  number  of 
which  definite  bacteria  have  been  satisfactorily  established  as 
casual.  Under  this  latter  class  we  now  have  such  prominent 
bacteria  as  Koch's  bacillus  of  tuberculosis,  Eberth's  bacillus  of 
typhoid  fever,  Klebs-Loffler's  bacillus  of  diphtheria,  Koch's  spiril- 
lum of  cholera,  Neisser's  diplococcus  (gonococcus)  of  gonorrhea, 


10  BACTERIOLOGY  : 

Nicolaier's  bacillus  of  tetanus,  Obermeier's  spirillum  of  relapsing 
fever,  and  Kitasato's  bacillus  of  plague.  Scarcely  less  impor- 
tant and  also  well  authenticated  is  the  bacillus  anthracis  of  malig- 
nant pustule,  bacillus  mallei  of  glanders,  bacillus  leprae  of 
leprosy  and  the  streptothrix  (?)  (ray  fungus)  of  actinomycosis, 
and  of  madura  foot.  To  these  should  be  added  the  well-recog- 
nized, micro-organismal  causes  of  many  septic  and  suppurative 
conditions — such  as  the  varieties  of  staphylococci,  so  frequent  in 
•abscesses;  the  streptococcus  found  in  erysipelas;  the  bacillus 
pyocyaneus  of  green  pus ;  the  bacillus  coli  communis,  frequent  in 
abscesses  adjacent  to  the  intestines ;  the  bacillus  aerogenes  eapsu- 
latus,  found  in  a  usually  fatal  form  of  sepsis  associated  with 
gaseous  edema,  etc. 

Nearly  all  of  these  organisms  have  been  proved  to  be  the 
specific  causes  of  their  respective  diseases  by  conforming  to  the 
well-known  rules  of  Koch.  That  is,  the  organism  is  found  to  be 
constantly  present  in  the  bodies  of  those  affected  by  the  disease, 
may  be  grown  in  pure  culture  outside  of  the  body,  when  properly 
inoculated  in  lower  animals  it  reproduces  the  disease,  and  may 
again  be  recovered  from  the  animal  so  infected. 

There  are  also  good  reasons  for  accepting  a  number  of  other 
organisms  as  specific.  Recent  investigations  corroborate  the 
claims  of  Sanarelli  that  his  bacillus  icteroides  is  the  cause  of  yel- 
low fever. 

The  bacillus  described  by  Canon  and  Pfieffer  for  influenza, 
and  by  Canon  and  Pielicke  for  measles,  are  generally  accepted. 
The  Lustgarten  bacillus  of  syphilis  has  not  been  sustained,  but 
van  Niessen  claims  to  -have  cultivated  from  syphilitic  blood  a 
bacillus  producing  characteristic  lesions  in  pigs  and  monkeys. 


ITS   PRACTICAL  VALUE  TO  THE   GENERAL   PRACTITIONER.    11 

The  claims  made  that  the  bacteria  causing  mumps,  scarlatina 
and  whooping-cough  have  been  isolated,  do  not,  as  yet,  appear 
conclusive;  while  the  organisms  of  such  notably  infectious  dis- 
eases as  typhus  and  small-pox  await  discovery.  The  better 
knowledge  of  the  bacteria  producing  the  infections  has  been  fruit- 
ful in  improving  hygienic  measures — a  subject  too  extensive  to  be 
considered  at  this  time.  Not  less  important  have  been  the  re- 
flections of  etiologic  advances  in  improvements  of  diagnosis, 
prophylaxis  and  treatment. 

Bacteriologic  Diagnosis. — Having  ascertained  that  a  micro- 
organism is  invariably  found  in  the  body  in  association  with  a 
single  disease  and  with  that  disease  only,  the  diagnosis  of  the  dis- 
ease may  readily  hinge  upon  the  determination  of  the  presence 
of  the  bacterium.  The  chief  methods  used  to  determine  the  char- 
acter of  u  micro-organism,  'and  thus  diagnose  the  disease,  depend 
upon:  (1)  Peculiarities  in  form  and  arrangement;  (2)  peculiari- 
ties of  staining;  (3)  peculiarities  of  cultural  growth;  (4)  pecul- 
iarities of  effects  produced  when  introduced  into  the  bodies  of 
certain  animals;  (5)  peculiarities  in  behavior  of  cultures  of  the 
micro-organism  when  brought  in  contact  with  certain  fluids  from 
the  diseased  animals. 

Certain  of  the  higther  forms  of  the  vegetable  parasites,  es- 
pecially the  parasitic  moulds  and  yeasts,  are  readily  recognized  by 
their  peculiarities  of  form  -alone.  For  example,  the  sacchar- 
omyces  <albicans  of  thrush  and  the  varieties  of  aspergilli  found  in 
the  external  ear  are  readily  determined  by  microscopically  ex- 
amining some  of  the  suspected  material  in  a  drop  of  -an  indiffer- 
ent fluid;  while  an  accurate  diagnosis  of  the  varieties  of  ring- 


12  BACTERIOLOGY  : 

worm,  of  favus,  of  tinea  versicolor,  may  be  obtained  by  removing 
some  of  the  affected  epithelial  scales  and  hairs  and  examining 
under  the  microscope  for  their  respective  moulds,  after  treating 
with  alcohol  and  ether,  and  strong  caustic  potash  to  remove  the 

fat. 

The  similarity  between  different  varieties  of  bacteria  is  so 
great,  however,  and  pathogenic  micro-organisms  are  so  frequent- 
ly aped  in  shape  and  grouping  by  the  benign  varieties,  that  this 
first  method  of  diagnosis,  as  a  rule,  is  merely  corroborative  to 
staining  and  cultural  methods.  Thus  the  most  noteworthy  pe- 
culiarity of  the  tubercle  bacilli  is  their  resistance  to  decolorizing 
agents ;  and  yet  it  is  also  very  important  that  the  organisms  ap- 
pear as  short  rods,  with  rounded  ends,  a  slight  curve  and  a  ten- 
dency to  be  arranged  in  II,  V  or  X  like  figures.  The  simple 
method  of  diagnosing  this  bacillus  by  staining  has  proved  of  chief 
value  in  tuberculosis  of  the  respiratory  tract,  the  presence  of 
tubercle  bacilli  in  the  sputum  being  practical  proof  of  the  ex- 
istence of  the  disease.  The  same  method  of  diagnosing  tuber- 
culosis in  other  parts  of  the  body  has  given  less  satisfaction.  In 
tuberculosis  of  the  genito-urinary  tract,  it  is  often  difficult  to  find 
the  bacilli  in  stained  preparations  of  urinary  sediment,  and  the 
bacilli  are  rarely  seen  in  the  pus  from  tuberculous  abscesses.  Al- 
though sputum  that  has  become  putrid  may  continue  to  show  the 
characteristic  bacilli,  it  is  advisable  to  make  preparations  of  fresh 
fluids.  Urine,  especially,  should  be  speedily  centrifuged  and  ex- 
amined, and  it  is  probably  an  advantage  to  use  a  method  which 
obviates  certain  of  the  extraneous  urinary  substances.  While  the 
smegma  bacillus  and  the  leprosy  bacillus  resemble  the  tubercle 


ITS   PEACTICAL  VALUE   TO   THE   GENERAL   PRACTITIONER.    13 

bacillus  in  staining  properties,,  it  is  to  be  recalled  that  both  of 
the  former  bacilli  stain  more  rapidly,  decolorize  more  easily  and 
have  different  grouping  tendencies  than  the  latter.  The  failure 
to  find  tubercle  bacilli,  of  course,  does  not  necessarily  negative 
the  presence  of  the  disease,  nor  may  the  number  of  bacilli  found 
bear  any  relation  to  the  extent  of  the  disease  process. 

Gonorrhea,  'at  present,  is  most  accurately  diagnosed  by  stain- 
ing. The  organism,  which  is  a  rather  large  diplococcus  with 
flattened  surfaces  in  opposition,  and  a  tendency  to  be  found  in 
groups  in  or  upon  the  cells,  is  differentiated  from  most  other 
diplococci  by  failing  to  stain  by  Gram's  method,  although  readily 
coloring  with  the  ordinary  basic  anilin  dyes.  In  subacute  or 
chronic  cases  the  gonococci,  having  invaded  the  deeper  epithelial 
layers,  may  be  absent  from  the  discharge  except  during  exacerba- 
tions. It  has,  therefore,  been  suggested  that  in  gleets  of  obscure 
nature  the  "Beer  Test"  be  used ;  the  visit  to  Bacchus  being  fre- 
quently rewarded  by  the  return  of  the  gonococcus  to  the  dis- 
charge. 

The  staining  of  cerebro-spinal  fluid  withdrawn  by  the  lumbar 
puncture  has  been  a  decided  aid  in  diagnosing  certain  varieties  of 
cerebro-spinal  inflammation.  The  diplococcus  intracellularis 
meningitidis  of  Weichselbaum,  frequent  in  the  epidemic  and  very 
fatal  form,  resembles  the  gonococcus  by  its  presence  in  the 
leucocytes  and  by  being  decolorized  by  Gram's  method.  The 
diplococcus  of  pneumonia  has  also  been  found  in  the  fluid  of 
many  of  the  fatal  cases. 

In  the  diagnosis  of  gastric  carcinoma  probably  the  most  sug- 
gestive addition  to  the  stomach  contents,  apart  from  the  presence 


^4  BACTERIOLOGY  : 

of  detached  bits  of  tumor,  is  the  long  immobile,  club-shaped 
Oppler-Boas  bacillus. 

The  most  important  recent  advance  in  the  diagnosis  of  puer- 
peral sepsis  is  the  employment  of  bacteriologic  methods  to  as- 
certain the  variety  of  the  infecting  organism.  The  fatal  infec- 
tions with  the  gas  bacillus  (bacillus  aerogenes  capsulatus  of 
Welch)  and  with  the  septicemia-producing  streptococcus  should 
be  carefully  separated  from  the  milder  infections  by  the  colon 
bacillus  and  the  staphylococcus  and  the  usually  localized 
gonococcus  invasions.  A  sterile  glass  tube  may  be  inserted  into 
the  uterine  cavity,  some  of  the  lochia  withdrawn  into  the  tube, 
and  the  tube  ends  sealed  pending  the  examination  of  the  contents. 

In  general  surgery  the  value  of  staining  the  fluids  from  in- 
fected foci,  even  during  the  operation,  in  order  to  determine  the 
necessary  operative  procedure,  has  not  diminished,  although  the 
method  is  not  new. 

Certain  bacteria  exhibit,  under  varying  conditions,  altera- 
tions in  their  staining  properties.  Such  bacteria  may  be  more 
distinctively  stained  after  cultivation  upon  a  particular  medium. 
This  is  so  important  with  the  diphtheria  bacillus  that  the  diag- 
nosis is  made  by  staining  after  cultivation  upon  Loffler  blood- 
serum  mixture.  The  rapid  growth,  at  incubator  temperature. 
of  the  irregularly  rounded,  elevated,  porcelain  white  colonies 
tending  to  rapidly  coalesce  into  a  diffuse  smeary  layer,  is  also 
quite  characteristic  of  this  organism.  Eisner,  Kashida  and  Hiss 
have  devised  special  media1  for  developing  characteristic  cultures 

1  Piorkowsky  (Ber.klin.  Woch.,  7,99,)  has  recently  recommended  a  medium 
composed  of  normal  urine  containing  0.5*  peptone  and  8  3*  gelatin.  The  medium  Is 
Inoculated  from  the  stools,  and  incubated  20  hours  The  typhoid  colonies  appear  aa 
colorless  spots  of  radiating  threads,  while  the  colon  cultures  are  sharply  defined, 
round,  yellowish  colonies. 


ITS  PEACTICAL   VALUE   TO   THE   GENERAL   PRACTITIONER.     15 

from  the  typhoid  bacillus,  but  their  practical  diagnostic  value  can 
hardly  compare  with  the  simple  method  of  serum  diagnosis.  Cul- 
tures, at  present,  are  of  chief  value  to  the  practitioner  in  diagnos- 
ing diphtheria,  and  those  who  have  not  the  advantage  of  a  labora- 
tory may  procure  the  necessary  media,,  and  with  a  little  training 
make  their  own  examinations.  In  lieu  of  an  incubator  the  inocu- 
lated tube,  in  a  protecting  case,  may  be  carried  in  an  inner  pocket, 
the  body  supplying  the  necessary  warmth. 

Animal  inoculations  have  proven  useful  in  diagnosing  genito- 
urinary tuberculosis  in  cases  where  the  bacilli  were  not  shown  by 
staining.  Some  of  the  fresh  urinary  sediment  (obtainable  from  a 
single  kidney  by  the  ureteral  catheter)  is  injected  into  the  ab- 
dominal wall  of  a  guinea  pig  that  is  killed  after  five  weeks  and 
examined  for  tuberculous  lesions.  Animal  inoculations  are  also 
of  value  in  diagnosing  the  type  of  many  of  the  infections ;  while 
the  subdural  inoculation,  in  a  rabbit,  of  a  bit  of  the  spinal  cord  of 
the  affected  animal  remains  the  best  method  of  ascertaining  the 
existence  of  rabies. 

One  of  the  greatest  advances  in  bacteriologic  diagnosis  is  that 
dependent  upon  the  tendency  of  the  bacteria  of  >a  given  disease  to 
agglutinate,  or  collect  in  clumps,  and  to  lose  their  motility  when 
brought  in  contact  with  the  serum  from  a  person  affected  by  the 
disease.  Although  this  method  is  useful  in  determining  the 
variety  of  the  bacteria,  the  presence  of  infective  organisms  in 
drinking  water,  etc.,  its  greatest  value  has  been  in  diagnosing 
disease. 

Being  present  in  over  ninety-five  per  cent,  of  cases  of  ty- 
phoid, it  is  the  most  useful  diagnostic  sign  yet  discovered.  The 


16  BACTERIOLOGY  : 

method  has  not  been  as  thoroughly  developed  in  other  diseases, 
but  has  shown  a  value  in  aiding  the  identification  of  cases  of 
cholera,  glanders,  malta  fever/  tuberculosis,  leprosy,  relapsing 
fever  and  other  diseases.  Considering  that  the  method  is  yet  in 
its  infancy,  the  results  obtained  have  been  surprisingly  accurate. 
The  test  is  performed  with  or  without  the  microscope,  the  former 
having  the  -advantage  of  quickness  and  greater  accuracy.  The 
main  difficulty  that  the  practitioner  will  find  with  this  method  is 
the  difficulty  of  keeping  on  hand  fresh  cultures  of  the  bacilli.  The 
advantages  of  emulsions  of  dead  bacilli  are  readily  apparent,  but 
thus  far  they  have  proven  less  reliable  than  the  fresh  cultures. 

A  final,  simple  and  practical  method  of  diagnosis  is  by  the  in- 
jection of  bacterial  products  into  the  affected  animal.  Tuber- 
culin, a  glycerin  extract  of  tubercle  bacilli,  has  proved  to  be  a 
most  reliable  agent  for  diagnosing  tuberculosis.  Unfortunately, 
its  tendency  to  exacerbate  this  affection  has  largely  precluded  its 
use  in  the  human  family.  Mallein  has  also  proven  successful  in 
diagnosing  glanders  in  horses. 

Prophylaxis  and  Treatment. — Two  main  classes  of  bacterial 
remedies  have  been  developed,  namely,  those  obtained  directly 
from  the  micro-organism  and  those  indirectly  obtained  by  the  ac- 
tion of  bacterial  products  upon  animals.  To  the  first  class  be- 
long the  toxins  -and  vaccines ;  to  the  latter  class  the  antitoxic  and 
bactericidal  serums.  The  former  <have  been  of  chief  value  as 
prophylactic  'and  immunizing  agents,  but,  as  yet,  have  not  re- 
vealed the  curative  powers  of  the  latter  class.  Upon  the  other 
hand,  the  antitoxins  have  more  transient  immunizing  powers 
than  the  toxins  or  vaccines.  Protective  inoculations  or  vaccin- 


ITS    PRACTICAL    VALUE    TO   THE    GENERAL   PRACTITIONER.    17 

ations  are  now  accessible  to  the  practitioner  for  small-pox,  an- 
thrax, cholera,  plague,  hydrophobia,  and  for  a  number  of  diseases 
of  the  lower  animals — as  black  leg  (symptomatic  anthrax),  hog 
cholera,  ttc. 

The  curative  effect  of  the  bacterial  products  of  the  tubercle 
bacillus  devised  by  Koch  have  hardly  proven  commensurate  with 
their  dangers,  while  the  employment  of  Coley's  mixed  toxins  of 
prodigiosus  and  erysipelas  for  the  relief  of  inoperable  malignant 
tumors  has  been  followed  by  a  cure  only  in  occasional  cases.  .The 
antitoxins  have  shown  temporary  but  valuable  immunizing 
powers  in  diphtheria  and  tetanus,  and  the  diphtheria  antitoxin 
has  revealed  marked  curative  powers — much  greater,  indeed,  than 
that  of  tetanus. 

The  use  of  streptococcus  antitoxin  has  been  followed  by  good 
results  in  certain  cases  of  sepsis  and  even  small-pox.  The  results 
suggest  the  desirability  of  greater  accuracy  in  diagnosing  the 
variety  of  the  infecting  organism  as  an  aid  to  the  picper  treat- 
ment. 

With  a  prospect  for  the  future  of  increased  and  simplified 
diagnostic  advances,  and  multiplied  and  more  efficient  curative 
products,  the  advantage  of  a  more  thorough  knowledge  of  bac- 
teriology to  the  practitioner  are  not  easily  overestimated. 


AN    ATLAS 

OF  THE 

BACTERIA   PATHOGENIC  IN  MAN. 


An  Atlas  of  the  Bacteria  Pathogenic  in 
the  Hitman  Subject. 

PART  I. 

BY   SAMUEL    G.    SHATTOCK,    F.R.C.S. 


THE  object  of  the  accompanying  plates  is  to  present  graph- 
ically from  original  preparations  all  the  chief  bacteria  which  are 
pathogenic  in  the  human  subject. 

The  originals  of  the  drawings  (in  the  Museum  of  the  Royal 
College  of  Surgeons,  London)  have  been  made  under  the  au- 
thor's supervision  by  Mr.  G,  T.  Gwilliam,  F.R.A.S.,  who,  from 
an  astronomical  experience,  is  a  draughtsman  of  extreme  accur- 
acy, and  they  may  be  relied  upon  as  absolutely  faithful  repre- 
sentations of  the  objects  themselves. 

All  the  preparations  have  been  drawn  as  viewed  under  a  ~ 
homogeneous  immersion,  Leitz,  ocular  No.  2,  tube  length  170 
millimeters,  giving  a  magnification  of  680.  It  is  to  be  noted, 
however,  that  in  order  not  to  tax  the  eye,  they  have  in  all  cases 
been  slightly  enlarged  in  the  drawing.  The  actual  magnifica- 
tion amounts  to  about  1,000. 

Each  illustration  gives  the  chief  forms  selected  from  several 
fields  in  order  to  bring  out  their  variations,  their  differences  in 
size,  or  their  grouping,  etc.,  a  result  which  will  dispel  the  idea 
of  uniformity  in  these  respects  that  is  sometimes  gained  from 
representations  where  such  differences  are  studiously  excluded. 

All  the  drawings  have  been  made  from  cultures  in  an  active 

21 


22  PATHOGENIC   BACTERIA 

stage  of  growth,  in  order  that  abnormal,  degenerate  forms  might 
be  eliminated.  The  precise  mode  of  preparation,  the  age  of  the 
culture,  and,  when  important,  its  original  source,  are  given  un- 
der the  several  figures. 

The  fungi,  which  are  pathogenic  in  the  human  subject,  be- 
long to  the  botanical  subdivision  of  (1)  Schizomycetes  (^zVctf, 
I  split,  KvHrjS,  a  fungus)  Fission-fungi,  or  Bacteria;  (2)  Blas- 
tomycetes  (BXaffroZ,  a  bud)  Budding-fungi,  or  yeasts;  (3)  Hy- 
phomycetes  ("TpoZ,  a  web),  Hyphal-fungi,  or  moulds. 

Strictly  speaking,  the  study  of  bacteriology  does  not  com- 
prise that  of  yeasts  and  moulds,  which  are  for  this  reason  ex- 
cluded from  some  systematic  works  devoted  to  bacteriology. 
Much  less  could  the  consideration  of  animal  micro -parasites  find 
a  place  in  a  strict  system  of  bacteriology,  although,  owing  to  its 
present  small  dimensions,  this  subject  is  included,  and  conven- 
iently so,  in  at  least  one  standard  English  work  on  "  Bacter- 
iology." 

With  the  exception  of  thrush,  all  the  illustrations  given  are 
limited  to  bacteria  proper  or  schizomycetes. 

In  regard  to  this  classification  of  pathogenic  fungi,*  it  will 
be  enough  to  say  that  bacteria  are  achlorophyllous  vegetable  or- 
ganisms, characterized  by  a  fissiparous  method  of  reproduction, 
though  in  a  certain  number  there  occurs  a  second  or  additional 
method,  viz.,  spore-formation. 

Yeasts,  whilst  again  they  may  multiply  by  spore-formation, 
do  so  as  an  ordinary  method  by  budding,  i.e.)  in  place  of  the 
cell  becoming  symmetrically  partitioned  into  two  or  more  ele- 

*  The  absence  of  chlorophyll,  which  has  commonly  served  as  a  basis  for  defining 
the  group  of  fungi,  is  not  held  by  Sachs  as  sufficiently  fundamental  for  such  a  sub- 
division; he  classes  with  achlorophyllous  schizomycetes  certain  chlorophyllous 
forms  which  multiply  by  a  similar  method,  under  the  name  of  schizophyta,  etc. 


OF   THE   HUMAN   SUBJECT.  23 

ments,  there  grow  out  one  or  more  processes  or  buds,  which, 
after  increasing  to  a  certain  size,  may  become  abstricted  off  and 
disconnected  from  the  parent  cell,  or  whilst  acquiring  an  inde- 
pendent cavity,  may  remain  connected  with  it  and  generate 
further  elements  by  the  same  plan.  The  hyphomycetes  occupy 
a  higher  position  than  either  of  the  foregoing  groups,  in  that 
they  present  distinct  organs  of  fructification,  and  are  capable, 
moreover,  of  a  proper  sexual  process  of  propagation. 

Not  that  these  subdivisions  are  more  clearly  defined  than 
are  others  in  biology. 

The  branching  filamentous  forms,  at  times  met  with  in  the 
tubercle  and  tetanus  bacillus,  make  an  approach  toward  the 
mycelial  productions  of  moulds;  and  in  actinomyces  and  strep- 
tothrix  Madurae,  a  dense  and  branched  mycelium  is  regularly 
developed  in  artificial  cultures. 

The  microscopic  characters  of  bacteria  are  in  scarcely  any 
case  sufficient  to  allow  of  their  identification.  It  is  by  the  en- 
semble of  their  cultural  characters  on  different  media,  their  chem- 
ical products,  the  results  of  experimental  inoculation  upon  ani- 
mals, that  identification  is  possible,  and  to  these  must  be  added 
the  effects  produced  upon  bacteria  by  the  action  of  specifically 
immunized  sera. 

Accumulated  observations  have  disclosed  a  bacterial  king- 
dom of  such  dimensions  that  to  determine  or  identify  any  given 
individual  is  a  matter  of  increasing  difficulty.  The  problem  of 
identification  is  reduced,  however,  within  comparatively  easy 
limits  when  those  organisms  which  are  pathogenic  are  alone  con- 
sidered, and  still  more  so  when  attention  is  restricted  to  those 
that  cause  disease  in  the  human  subject. 

The  fundamental  argument  adopted  by  Darwin  for  the 


24  PATHOGENIC   BACTERIA 

theory  of  evolution,  viz.,  the  impossibility  of  defining  species, 
holds  equally  through  microscopic  and  macroscopic  forms  of  life. 
Nevertheless,  of  the  different  methods  of  classifying  bacteria, 
those  founded  upon  form  are  the  only  ones  at  present  practica- 
ble. Of  such  classifications  there  are  almost  as  many  as  there 
are  authors.  For  all  practical  purposes,  however,  the  patho- 
genic bacteria  are  reducible  to  spheres  and  rods;  the  latter  being 
of  the  most  various  lengths,  at  times  produced  into  long  fila- 
ments, which  may  be  straight  or  spirally  twisted,  or,  in  a  few 
instances,  branched. 

As  to  the  terms  bacterium  and  bacillus,  the  former  is  now 
obsolete;  every  straight,  rod-shaped  organism  is  a  bacillus.  If 
the  first  is  still  employed,  it  is  only  in  deference  to  pre-estab- 
lished usage;  the  early  distinction  between  the  two,  founded  as 
it  was  upon  difference  in  length,  is  rendered  valueless  by  the  var- 
iations in  this  respect  presented  by  one  and  the  same  organism; 
in  the  more  highly  pleomorphic  examples,  the  shortest  forms  of 
bacilli  may  be  indistinguishable  from  spheres  or  cocci.  The  pro- 
posal to  name  as  "bacilli"  straight,  rod-like  organisms  which 
form  spores,  and  "bacteria"  those  that  do  not,  has  not  been 
followed,  since  there  is  nothing  whatever  in  the  terms  indicative 
of  such  a  difference.  Besides  the  straight  rod  or  bacillus,  the 
other  chief  forms  of  pathogenic  bacteria  are  the  spherical,  or 
cocci;  and  the  twisted  rod  or  spirillum. 

It  may  be  observed,  in  passing,  that  micrococci  are  not  in 
all  cases  geometrically  spherical.  The  faces  of  subdivision  are 
very  frequently  flattened  so  long  as  the  resulting  elements  re- 
main united,  whatever  disposition  the  grouping  takes — whether 
in  twos  (diplococci),  fours  (tetracocci),  eights  (sarcinacocci),  or 
linear  series  (streptococci);  it  is  not  rare  again  to  find  the  ele- 


OF   THE   HUMAN   SUBJECT.  25 

ments  of  a  chain — or  streptococcus  elongated  in  the  direction  of 
its  length,  i.e.,  oval  in  place  of  spherical. 

The  microscopical  examination  of  bacterial  cultures  may  be 
reduced  to  a  comparatively  simple  technique.  Bacteria  may  be 
examined  after  being  dried,  or  in  their  natural  condition.  The 
advantage  of  the  first  method  is  that  the  preparations  are  perma- 
nent, and  to  this  must  be  added  the  greater  facility  with  which 
they  admit  of  being  drawn  or  photographed.  Under  the  other, 
the  organisms  suffer  no  reduction  in  size  or  alteration  in  form, 
nor  can  appearances  artificially  produced  be  mistaken  for  such  as 
are  natural.  There  is  the  same  difference,  in  short,  as  between 
the  study  of  plants  growing  in  a  garden  and  those  dried  in  a 
herbarium.  The  second  method  is,  therefore,  in  all  cases  pref- 
erable; obviously  for  the  study  of  growth  and  reproduction  it  is 
absolutely  necessary. 

The  Hanging  Drop. — Examinations  in  the  natural  state  are 
carried  out  by  means  of  the  hanging  drop,  which  may  be  de- 
scribed in  detail  as  being  the  most  beautiful  of  all  methods,  and 
as  expeditious  as  any. 

If  a  culture  on  agar  or  gelatine  (when  this  is  not  liquefied 
by  the  growth)  is  to  be  examined,  a  thin,  narrow  ring  of  vaseline 
is  painted  around,  but  a  short  way  from  the  depression  of  a  hol- 
low ground  slide;  a  single  6'se  or  loop  of  distilled  water  is  then 
transferred  to  the  centre  of  a  square  or  circular  cover-glass  (No. 
1  thickness).  For  the  sake  of  those  unacquainted  with  such 
matters,  it  may  be  said  that  the  6'se  or  loop  is  a  small  eyelet 
made  at  one  end  of  a  piece  of  platinum  wire  about  two  and  a 
half  inches  in  length,  the  other  end  of  which  is  pressed  into  the 
end  of  a  piece  of  solid  glass  rod,  seven  inches  long,  softened  in  the 
flame  of  a  Bunsen  burner;  the  diameter  of  the  eyelet,  which  is 


26  PATHOGENIC   BACTERIA 

made  by  bending  the  wire  with  the  end  of  a  small-pointed  pair 
of  forceps  into  a  complete  circle,  should  be  two  millimeters 
(about  ^  inch),  and  in  all  cases  it  is  to  be  sterilized  by  raising  it 
to  a  red  heat  in  the  flame  before  and  after  use  (Fig.  I).  After 


Fig.  1.— The  6se  or  platinum  loop,  the  length  of  the  wire  and  glass  rod  reduced. 

sterilization  in  the  flame,  the  loop  is  inserted  into  the  culture 
tube,  applied  to  the  growing  edge  of  the  culture,  withdrawn, 
and  immediately  transferred  to  the  drop  of  water  on  the  cover- 
glass,  in  which  it  is  moved  until  slight  turbidity  appears;  the 
excess  of  the  culture  on  the  loop  is  now  burnt  off  in  the  flame, 
and  the  bacteria  thoroughly  distributed  on  the  cover-glass  by  its 
means,  the  drop  being  extended  so  as  to  cover  an  area  of  about 
four  millimeters  in  diameter.  The  cover  glass  is  then  seized  at 
the  edge  in  a  small  pair  of  forceps,  turned  over,  and  applied  to 
the  hollow  ground  slide  so  that  the  minute  drop  of  fluid  lies 
over  the  center  of  the  hollow  space  in  the  slide  without  coming 
into  contact  with  the  latter;  the  gentlest  pressure  made  around 
the  border  of  the  cover-glass  will  then  render  the  moist  chamber 
completely  air-tight  (Fig.  2).  A  moist  chamber  may  be  impro- 


Fig.  2.— Section  of  hollow  ground    slide  with  cover-glass  in  situ,  slightly  re- 
duced. 

vised,  however,  in  a  very  effective  manner  on  an  ordinary  flat 
slide,  as  follows:  Cut  a  piece  of  thick  blotting-paper  an  inch 
square,  fold  it,  and  cut  away  a  small  semi-circle  from  the  center 
of  the  folded  border,  open  it  out,  wet  it,  and  lay  it  on  the  slide; 


OF   THE   HUMAST   SUBJECT.  27 

the  cover  glass  having  been  prepared  exactly  as  before  is  inverted 
so  that  the  hanging  drop  lies  over  the  centre  of  the  circular  hole 
in  the  blotting  paper  (Fig.  3). 


Fig.  3.— Section  of  slide  and  moist  chamber  prepared  with  blotting-paper 
slightly  reduced. 

If  the  examination  be  prolonged,  it  is  necessary  to  moisten 
the  paper  from  time  to  time.  For  the  study  of  such  prepara- 
tions, much  of  the  light  must  be  cut  off  with  the  diaphragm, 
assuming  that  the  Abbe  condenser  and  plane  side  of  the  mirror 
are  used;  unstained  organisms  are  otherwise  scarcely  discernible. 

The  most  suitable  area  for  observation  is  the  edge  of  the 
drop  where  the  film  of  fluid  is  thin,  and  where  there  is  what  may 
be  called  a  "still"  layer  in  which  an  accumulation  of  the  bac- 
teria takes  place,  and  where  their  movements  (whether  active  or 
Brownian)  are  less  lively,  and  eventually  cease. 

The  best  plan  of  finding  the  edge  is  to  place  the  slide  with 
the  hanging  drop  centrally  on  the  stage  of  the  microscope, 
transfer  a  droplet  of  cedar  oil  exactly  over  the  hanging  drop, 
and  screw  down  the  coarse  adjustment  until  the  oil  rises  up  to 
meet  the  object  glass  ;  the  rest  of  the  focussing  is  then  carried 
out  by  means  of  the  fine  adjustment  which  is  screwed  until 
some  of  the  bacteria  come  into  focus  ;  the  slide  is  then  moved  in 
any  one  definite  direction  until  the  edge  of  the  drop  is  reached. 
Besides  the  forms,  the  motility  of  the  bacteria  is  to  be  deter- 
mined by  the  hanging  drop.  For  the  latter  purpose,  the  use  of 
broth  cultures  is  preferable  to  those  made  on  agar ;  nevertheless, 


V?8  PATHOGENIC   BACTERIA 

if  a  preparation  of  the  typhoid  bacillus,  e.g.,  is  made  from  an 
agar  culture  of  twenty-four  hours'  growth,  in  the  way  described, 
there  is  no  difficulty  in  observing  the  inotility  of  the  microbe, 
though  the  movements  are  not  so  active  as  in  a  broth  culture. 
Examined  in  the  hanging  drop,  it  is  to  be  noted  that  even  non- 
motile  bacteria  present  marked  passive  or  Brownian  move- 
ments ;  these  are  distinguishable  by  the  fact  that  no  real  loco- 
motion takes  place,  the  movements  being  of  a  to-and-fro  or 
oscillatory  character. 

Coloration  of  the  Hanging  Drop. — The  hanging  drop  having 
been  so  viewed,  may  next  be  advantageously  stained.  It  is  not 
necessary  for  this  purpose  to  make  a  new  preparation.  The 
cover-glass  is  removed  and  turned  over  on  a  piece  of  filter  paper, 
so  that  the  drop  is  again  brought  uppermost. 

The  best  staining  fluid  is  a  dilute  solution  of  gentian  violet, 
made  by  adding  .5  c.c.  of  an  aqueous  solution  of  gentian  violet 
(gentian  violet  2.25  grammes,  distilled  water  100  c.c.)  to  10 
cubic  centimeters  of  distilled  water.  The  dilution  may  be  ar- 
rived at  sufficiently  well  by  placing  a  few  drops  of  the  gentian 
violet  solution  in  a  test  tube  and  adding  distilled  water  until  the 
fluid  is  sufficiently  dilute  to  allow  of  being  seen  through  when 
held  between  the  eye  and  the  light.  The  sterilized  6se  is  dipped 
into  this,  and  the  drop  of  dye  mixed  on  the  cover- glass  with  the 
hanging  drop  already  there  ;  the  cover-glass  is  then  again  in- 
verted and  replaced  over  the  hollow  slide  or  upon  the  blotting- 
paper  of  the  moist  chamber.  The  ordinary  histological  method 
of  running  in  the  dye  from  one  side  of  a  wet  preparation  made 
without  hollow  slide  or  moist  chamber,  is  followed  by  too  much 
displacement. 

The  action  of  the  dye  is  easily  studied  in  all  its  grades  in 


PLATE  XXIII 


s 


>4/6/ca/»s. 


MEDICAL    ANNUAL,    1898 


PLATE  XXIM. 

SACCHAROMYGES  ALBIOANS.    THE  BLASTOMYCES  OR  YEAST-CAUSING  THRUSH. 

The  drawing  is  made  from  a  hanging  drop  of  a  broth  culture  which  had  been 
incubated  at  37*  C.  for  24  hours. 

There  is  shown  on  the  left  and  upper  part  of  the  figure  a  series  of  simple  oval  cells  ; 
and  below  these,  similar  cells  from  which  the  formation  of  a  second  element  is  pro- 
ceeding ;  this  appears  as  a  minute  bud  growing  out  from  one  of  the  ends  or  side  of  the 
parent  cell.  Where  the  cells  lie  in  groups  their  opposed  faces  are  flattened  or  facetted, 
an  extensive  group  resembling  a  mosaic.  The  process  whereby  a  filamentous  stage  is 
reached  consists  in  the  elongation  of  the  cells,  which  continue  to  give  rise  to  others 
from  apex  and  sides  until  a  complex  branching  system  results.  The  chief  secondary 
branches  arise  from  the  nodes  of  the  main  filaments,  and  from  the  former  a  tertiary 
set  of  smaller  cells,  and  so  forth. 

On  the  right  hand  side  are  shown  four  cells,  the  protoplasm  of  which  has  been  tinted 
with  a  dilute  aqueous  solution  of  gentian  violet ;  they  exhibit  a  certain  number  of 
spaces  filled  with  fluid,  or  vacuolea,  analogous  to  those  holding  sap  in  higher  plants  ; 
and  spherical  granules,  the  function  of  which  is  not  yet  known,— possibly  zymogenic 
in  nature. 

In  the  elongated  or  filamentous  cells  the  vacuoles  are  sometimes  of  considerable 
length,  and  can  be  readily  seen  in  tbe  unstained  condition. 


PLATE  XXIV 


Fig.  1.— Typhoid  Bacillus. 


Fig.  2.— Typhoid  Bacilli. 


MEDICAL    ANNUAL,    1898 


PLATE   XXIV. 

FIG  1.— TYPHOID  BACILLUS. 

Prom  a  culture  24  hours'  old,  incubated  at  87°  C.,  and  made  on  the  surface  of  agar. 
Stained  with  carbol  fuchsin,  washed  in  water  acidulated  with  acetic  acid.     Straight  or 
very  slightly  curved  rods,  the  shortest  of  which  appear  as  oval :   the  bacilli  present 
great  variations  in  length  and  breadth:    parallel  grouping  is  fairly  common.    The 
filamentous  forms  exhibit  no  trace  of  a  segmented  or  composite  structure. 


FIG.  2. — TYPHOID  BACILLI. 

Five  typhoid  bacilli,  from  a  dried  preparation  stained  by  Van  Ermengen's  silver 
method,  and  showing  the  long  wavy  flagella  arising  around  them.  The  largest  number 
shown  is  seventeen,  but  this  may  be  exceeded. 


PLATE  XXV 


Fig.  1.— Colon  Bacillus. 


Fig.  2.  Colon  Bacillus. 


MEDICAL    ANNUAL,    i8(>8 


PLATE    XXV. 
FIG.   1. — COLON  BACILLUS. 

From  a  culture  in  a  simple  aqueous  solutiou  of  Peptone,  24  hours'  growth,  incubated 
tit  37  G. 

The  figure  is  introduced  to  show  the  "end-staining"  often  observed  in  bacilli,  the 
causation  of  which  is  not  fully  understood.  The  interest  attaching  to  the  present 
specimen,  is  that  it  demonstrates  the  effect  of  one  particular  factor  in  leading  to  this 
result.  It  has  been  noticed  by  Mr.  H.  C.  Haslam  that  the  "end-staining"  occurs 
regularly  in  the  colon  bacillus  if  it  is  grown  in  alkaline  media. 

In  an  unstained  hanging  drop  corresponding  appearances  present  themselves,  the 
ends  of  the  bacilli  being  darker  and  more  granular  than  the  centres. 


FIG.  2. — COLON  BACILLUS. 

From  an  agar  culture  of  48  hours'  growth,  incubated  at  37°  C. 

Showing  the  usual  form  of  the  Colon  Bacillus :  plump  straight  rods  with  rounded 
ends,  variable  in  length  and  thickness,  the  shortest  appealing  as  oval.  Parallel 
grouping  occurs.  As  there  are  many  varieties  of  Colon  Bacillus,  it  is  necessary  to  take 
one  as  a  standard  ;  that  selected  is  from  a  culture  of  Professor  Bscherich's  (the 
original  describer  of  the  bacillus).  A  subculture  of  this  particular  organism  was 
obtained  from  Professor  Bscherich  by  Mr.  Wallis  Stoddart,  to  whom  the  author  is 
indebted  for  a  sample  of  the  strain. 


PLATE  XXVI 


Fig.  1. — Staphylococcus  Pyogenes  Aureus. 


Fig.  2.— Plague  Bacillus. 


MEDICAL    ANNUAL,    i898 


PLATE  XXVI. 
FIG.  1. — STAPHYLOCOCCUS  PYOGENES  AUREUS. 

From  the  edge  of  a  growing  culture  on  agar. 

Carbol  fuchsin,  washed  in  acidulated  water.  The  cocci  occur  in  clusters  or 
botryoidal  groups  (2ra<J>vA»?-  bunch  of  grapes),  here  and  there  in  short  chains, 
twos,  or  singly.  The  individuals  in  the  groups  vary  in  size,  indicating  probably 
differences  in  age  ;  in  some  of  the  groups  the  process  of  division  appears  in  a 
diplococcus  with  flattened  sides,  the  elements  of  which  have  not  yet  parted  and 
become  spherical. 


FIG.  3— PLAGUE  BACILLUS. 

Agar  culture,  incubated  37°  C.;  the  advancing  edge  of  a  three  days'  growth. 

Carbol  fuchsin,  washed  in  acidulated  water.  Short  rods,  mostly  of  oval  form, 
often  in  pairs,  side  by  side.  Here  and  there  slightly  curved,  longer  forms  occur, 
some  of  remarkable  thickness. 

The  original  source  of  the  bacillus  was  a  Lascar  admitted  into  the  Seamen's 
Hospital,  Albert  Dock,  from  a  P.  and  O.  steamer  arriving  from  Bombay;  the 
bacteriological  diagnosis  was  made  by  Dr.  R.  T.  Hewlett;  inoculations  carried 
out  upon  guinea  pigs  caused  d^ath  with  typical  symptoms. 

The  forms  examined  in  the  blood  of  animals  experimentally  inoculated  with 
this  particular  bacillus  showed  marked  "end-staining";  this  is  entirely  absent 
in  the  specimen  prepared  from  the  agar  culture. 


PLATE  XXVII. 

GONOCOCCUS. 

A  cover  glass  preparation  of  urethral  pus,  made  by  means  of  the  loop,  from 
the  ureihral  meatus  in  a  case  two  days  after  infection. 

Carbol  fuchsin,  washed  in  acidulated  water.  Two  flattened  epithelial  cells 
from  the  urethra,  and  one  "  polynuelear  "  leucocyte  are  represented.  The  stain 
has  tinted  the  bodies  of  the  epithelial  cells,  but  not  that  of  the  pus  cell.  The 
cocci,  without  exception,  occur  in  pairs  with  flattened  or  slightly  concave  faces; 
here  and  there  is  a  group  of  four.  The  diplococci  are  set  in  groups  or  colonies 
which  lie  outside  and  unconnected  with  the  cells;  elsewhere,  upon  and  within 
them.  The  groups  connected  with  the  epithelial  cells  are  surrounded  with  an 
uncolored  zone  of  ground  substance  indicating  their  location  within  the  cell 
protoplasm.  Those  about  the  nucleus  of  the  leucocyte  are  in  cell  protoplasm. 


PLATE  XXVtt 


Gonococcus. 


MEDICAL    ANNUAL,    16 


PLATE   XXVIII. 
Fio.  1. — ANTHRAX  BACILLUS. 

Agar  culture,  incubated  at  87°  C.;  of  24  hours'  age  :  the  films  treated  with  20  per 
cent,  acetic  acid  before  staining  with  aqueous  solution  of  gentian  violet. 

The  lowest  filament  is  as  yet  sporeless  ;  some  of  its  component  segments  are  but  half 
the  length  of  others,  and  separated  by  a  narrower  interval,  indicating  that  fission  and 
interstitial  elongation  are  proceeding  in  the  filament. 

In  the  others,  various  stages  of  spore-formation  are  shown.  The  spores  appear  at 
first  as  minute  unstained  points  at  or  near  the  centre  of  the  segments,  the  capsule  of 
tha  spores  preventing  the  penetration  of  the  dye.  When  fully  grown,  the  spores  dis- 
tend the  wall  or  "sheath"  of  the  bacillary  cell,  from  which  they  are  subsequently  set 
free,  as  shown  at  the  highest  part  of  the  figure.  In  many  of  the  sporulating  segments 
there  is  a  minute  second  point,  probably  an  abortive  second  spore,  since  the  fully 
developed  spore  in  in  most  instances  not  strictly  central,  but  nearer  one  end  of  the 
spore-bearing  segment,  and  this  whether  the  latter  presents  a  second  point  or  not. 

The  staining  or  the  spores  is  best  carried  out  by  means  of  hot  carbol  fuchsin,  and 
the  use  of  acid  and  methylene  blue  as  described  under  the  Tubercle  bacillus. 


FIG.  2. — ANTHBAX  BACILLUS. 

Broth  culture  of  24  hours'  growth,  incubated  at  87°  C.  Carbol  fuchsin,  washed  in 
acidulated  water. 

Very  few  spores  were  found  in  the  preparations,  and  none  are  present  in  the 
filaments  figured.  The  spore  formation  takes  place  only  under  the  free  access  of  gaseous 
or  atmospheric  oxygen.  The  filaments  adhere  in  tresses  or  strands. 

Active  subdivision  is  taking  place  in  the  segments,  the  ends  of  which  remain  straight 
or  squarely  cut. 


PLATE   XXVUl. 


\  \ 


\ 


Fig.  2.— Anthrax  Bacillus. 

MEDICAL    ANNUAL, 


PLATE  XXIX. 
ANTHRAX  BACILLUS. 

,*™^eSS*ri   PreP"?.4'011"  of  a  small  colony  on  agar  in  a  Petri   capsule;   lowly 
magnified  ;  the  actual  diameter  of  the  colony  is  one  millimetre. 

Aqueous  solution  of  gentian  violet,  washed  in  acidulated  water 
or  ha1ry0p0rocelse?Stl  °f  *  feltw°rk  °f  wavy  fllamen<*>  extending  at  the  margins  in  tufts 

allowing  the  latter  to  fal1  on  the  colo»>- 


PLATE  XXIX 


Anthrax  Bacillus. 


MEDICAL    ANNUAL,    1898 


PLATE    XXX. 

TUBERCLE  BACILLUS. 

A  preparation  of  Tubercular  sputum,  distributed  over  the  cover-glass  in  the  fresh 
state.  Carbol  fuchsin,  heated  on  the  cover,  the  latter  then  transferred  to  a  watch 
glass  of  25  per  cent,  hydric  sulphate  till  decolourized,  washed  in  water,  and  then 
counter-stained  with  aqueous  solution  of  inethylene  blue  in  the  cold,  washed  again  in 
water,  and  allowed  to  dry.  Tubercular  sputum  is  best  examined  when  no  antiseptics 
have  been  used  for  its-  disinfection.  It  should  be  spread  out  in  a  thin  layer  over  a 
sheet  of  glass  on  a  black  surface,  or  one  of  the  black-bottomed  plates  made  for  the 
purpose;  the  most  purulent  foci  are  then  isolated  with  needle  and  forceps,  or  cut  out 
with  scissors  and  forcibly  broken  up  and  distributed  on  a  series  of  cover-glasses  with 
the  back  of  the  forceps,  whilst  the  covers  are  steadied  by  the  pressure  of  a  needle. 
Passing  the  films  through  the  name  is  not  necessary.  If  the  cover-glasses  are  not 
heated  after  the  carbol  fuchsin  has  been  filtered  on  to  them,  the  dye  must  be  allowed  to 
act  for  a  considerably  longer  time— fifteen  minutes:  if  heated,  they  must  be  held  over 
a  low  flame  in  a  small  pair  of  forceps  of  which  the  ends  have  been  somewhat  sharply 
bent,  in  order  that  the  dye  may  not  be  conducted  to  the  under  side  of  the  glass. 

The  rationale  of  this  particular  method  is  as  follows:— the  carbol  fuchsin  stains  all 
the  different  microbes  present  ia  such  a  film ;  with  the  exception  of  the  tubercle 
bacilli,  all  are  afterwards  decolourized  by  the  action  of  the  hydric  sulphate,  to  be 
re-dyed,  together  with  the  cell  nuclei,  by  the  methylene  blue.  This  peculiar  resistance 
to  the  action  of  the  acid  is  common  to  the  tubercle,  leprosy,  and  smegraa  bacilli. 

In  the  examination  of  pure  cultures  of  the  bacillus,  this  differential  method  is 
unnecessary,  and  the  organism  may  be  stained  simply  with  carbol  fuchsin,  or  by 
Gram's  method,  etc. 

In  addition  to  the  "  polynuclear"  leucocytes  (pus  cells),  there  are  shown  in  the 
figure  two  squamous  epithelial  cells  derived  from  the  upper  part  of  the  respiratory 
passages  ;  the  body  of  these  is  faintly  tinted  with  the  blue. 

Many  of  the  tubercle  bacilli  occur  in  small  grotesque  groups,  that  have  been 
likened  to  Chinese  characters. 

The  bacilli  are  mostly  slightly  curved,  their  extremities  rounded,  and  their  proto- 
plasm segmented,  so  that  the  microbes  appear  "  beaded."  The  beading  is  not  invariable 
in  such  preparations  of  sputum. 


PLATE  XXX 


Tubercle  Bacillus. 


MEDICAL    ANNUAL,    2898 


OF  THE   HUMAN   SUBJECT.  47 

such  a  hanging  drop,  motile  bacteria  retaining  their  power  of 
movement  whilst  the  coloration  is  proceeding  in  them. 

To  Make  a  Permanent  Preparation  of  the  Hanging  Drop. — 
Lastly,  a  permanent  specimen  may  be  made  from  the  same  prep- 
aration by  removing  the  cover-glass,  placing  it  again  with  the 
drop  upwards  on  a  piece  of  filter  paper  beneath  a  watch-glass  or 
bell-jar,  and  allowing  it  to  dry  ;  after  which  a  small  drop  of 
Canada  balsam  (dissolved  in  xylol)  is  placed  on  a  slide,  a  second 
small  drop  on  the  dried  film,  and  the  latter  then  allowed  to  fall 
on  the  slide  in  the  usual  manner.  Or,  after  the  examination  of 
an  unstained  hanging  drop,  this  may  be  allowed  to  dry  in  the 
same  way  and  treated  in  the  manner  to  be  presently  described 
under  the  dry  method. 

The  dyeing  of  hanging  drops  made  from  broth  cultures  (in 
which  case,  of  course,  no  water  need  be  placed  on  the  cover  - 
glass)  is  not  quite  so  satisfactory  as  those  from  agar,  in  conse- 
quence of  an  extremely  fine  colored  precipitate  appearing  in  the 
fluid,  probably  a  proteid  present  in  the  broth,  which  unites  with 
the  dye.  This  precipitate  is  unconnected  with  the  growth  of 
the  bacteria,  as  it  appears  if  sterile  broth  is  mixed  with  the 
diluted  dye  and  examined  as  a  hanging  drop.  Nevertheless,  in 
such  broth  preparations  when  colored,  many  fields  may  be  found 
devoid  of  adventitious  granules,  which,  it  may  be  noted  in  pass- 
ing, exhibit  lively  Brownian  movements. 

The  examination  of  jelly  that  is  being  liquefied  by  the 
growth  of  an  organism  is  conducted  precisely  as  that  of  a  broth 
culture ;  and  admirable  preparations  may  be  obtained  by  color- 
ing the  hanging  drop  with  dilute  solution  of  gentian  violet  in 
the  manner  already  described. 

Of  all  the  methods  of  bacterial  microscopy,   the  hanging 


48  PATHOGENIC   BACTERIA 

drop,  with  few  exceptions,  is  the  best ;  the  important  method  of 
Gram,  for  instance,  cannot  be  carried  out  except  on  a  dry  prep- 
aration. 

The  Dry  Method. — The  more  minute  attention  paid  to  de- 
tails, the  more  satisfactory  will  be  the  results. 

To  commence  with,  then,  the  cover-glasses  (No.  1  thick- 
ness) must  be  properly  clean.  They  are  to  be  moved  about  in  a 
capsule  or  other  flat  vessel  of  hydrochloric  acid,  washed  in  com- 
mon water  and  then  kept  in  a  wide-mouthed  stoppered  bottle  of 
absolute  alcohol,  to  which  glacial  acetic  acid  has  been  added  in 
the  proportion  of  10  per  cent.  The  addition  of  acid  will  be 
found  to  prevent  the  cover-glasses  receiving  too  high  a  polish 
when  wiped,  a  result  which  greatly  interferes  with  the  equable 
distribution  of  the  bacteria  over  the  surface.  Three  cover-glasses 
having  been  wiped  dry,  a  loop  of  distilled  water  is  placed  on 
each  ;  the  loop  should  be  drawn  a  short  way  over  the  surface, 
and  if  it  is  found  that  the  water  does  not  adhere  to  the  glass, 
but  returns  again  to  a  bead,  the  cover  must  be  replaced  in  the 
acidulated  alcohol  and  wiped  afresh.  The  sterilized  loop  having 
been  inserted  into  the  culture  tube  and  a  small  quantity  of  the 
growing  edge  withdrawn,  it  is  applied  to  the  drops  of  water  in 
succession,  and  the  residue  having  been  burned  off  in  the  flame, 
the  loop  is  used  to  distribute  the  turbid  drop  over  the  entire 
face  of  each  of  the  cover-glasses,  which  are  then  allowed  to  dry; 
the  distribution  is  facilitated  by  breathing  on  the  surface,  and 
the  glasses  are  best  steadied  by  the  pressure  of  a  needle.  The 
prepared  side  is  to  be  kept  upwards  throughout  all  the  subse- 
quent manipulations.  The  time-honored  custom  is  now  to  pass 
the  covers  thrice  across  the  flame,  with  the  object  of  coagulating 
the  albumen  and  fixing  the  films.  Although  so  generally 


OF   THE   HUMAN"   SUBJECT.  49 

adopted,  it  is  in  reality  wholly  unnecessary  ;  if  the  transit 
through  the  flame  is  made  sufficiently  slowly  to  really  heat  the 
glass,  there  is  a  risk  of  inducing  still  further  shrinkage  of  the 
bacteria.  Not  only  is  the  practice  unnecessary  for  preparations 
made  from  agar  cultures,  but  from  careful  comparative  experi- 
ments, the  author  has  found  it  equally  without  advantage  in  the 
case  of  such  as  are  made  from  liquefied  gelatine  or  from  broth  ; 
the  film  will  remain  adherent  even  if  the  dye  has  to  be  heated 
on  the  glass,  provided  always  that  it  is  not  afterwards  violently 
washed  beneath  a  stream,  but  in  a  vessel,  of  water  ;  to  such  cul- 
tures the  addition  of  water  is  not  required  ;  the  loop  is  simply 
dipped  into  the  fluid  and  distributed  over  the  cover-glass. 

The  next  procedure  is  that  of  staining  For  this  purpose 
aniline  dyes  are  alone  used  ;  none  others  are  sufficiently  intense. 
The  three  chief  are  carbol  fuchsin,  aqueous  solution  of  gentian 
violet,  and  aqueous  solution  of  methylene  blue  ;  for  special  pur- 
poses  modifications  are  necessary,  the  most  important  of  which 
are  Gram's  method  and  those  for  staining  spores,  capsules,  and 
flagella.  Of  these  dyes  carbol  fuchsin*  holds  the  first  place,  and 
takes  the  same  position  for  bacterial  staining  that  hematoxylin 
does  for  histological  :  it  not  only  dyes  all  known  bacteria,  but 
gives,  as  a  rule,  cleaner  preparations,  in  consequence  of  its  being 
more  readily  washed  out  from  extraneous  material. 

The  cover-glass  should  be  quite  covered  with  a  high  bead  of 
the  stain.  This  may  be  done  from  a  drop  bottle,  but  the  author 
invariably  filters  the  stain  directly  on  to  the  cover-glass  through 
a  small  cone  of  filter-paper  held  across  its  overlapping  parts  in  a 
pair  of  forceps.  The  stain  having  remained  on  from  five  to  fif- 

*  Fuchsin,    1    gram:   absolute    alcohol,  10  c.c.    Dissolve  and  add  lOOc.c.  of  a  5 
per  cent,  aqueous  solution  of  carbolic  acid. 


50  PATHOGENIC    BACTERIA 

teen  minutes,  the  cover-glass  is  taken  up  with  a  fine  pair  of 
pointed  forceps,  tilted  to  throw  off  the  stain,  and  at  once  gently 
submerged  in  a  saucer  of  water  faintly  acidulated  with  acetic 
acid  ;  the  washing  may  be  completed  in  a  second  or  a  third 
saucer,  and  finally  in  unacidulated  water.  The  degree  of  acidity 
can  be  arrived  at  by  pouring  a  few  drops  of  glacial  acetic  acid 
into  a  saucer,  inverting  it  to  get  rid  of  the  excess,  and  then  fill- 
ing with  water,  common  or  distilled — a  solution  of  about  .5  c.c. 
glacial  acetic  acid  to  70  c.c.  of  water. 

The  washing  is  preferably  carried  out  in  this  way  rather 
than  beneath  a  stream,  as  the  latter  tends  to  displace  the  film  ; 
nor  is  the  excess  of  the  dye  so  well  removed  by  water  without 
the  acid. 


Fig.  4.— Sheet  of  lead  bent  at  right  angles,  with  filter-paper  folded  upon  it,  and 
cover-glass  stood  edgewise  to  drain  without  the  film  coming  in  contact  with  the 
paper. 

After  the  final  washing  the  cover-glass  is  allowed  to  drain 
and  to  dry  by  placing  it  obliquely  with  the  prepared  surface 
downwards,  against  a  sheet  of  filter-paper  folded  at  right  angles 
over  a  piece  of  lead  (Fig.  4).  It  may  be  more  rapidly  dried  by 
pressing  between  filter-paper,  but  at  the  risk  of  damaging  the 
film  and  transferring  fibers  to  it.  When  dry,  the  preparation  is 
completed  by  mounting  it  on  a  slide  with  a  xylol  solution  of 
Canada  balsam. 

In  a  certain  few  cases  (as  in  agar  cultures  of  the  anthrax 


OF  THE   HUAIAH   SUBJECT.  51 

bacillus)  this  method  does  not  satisfactorily  clear  the  prepara- 
tion, i.e.,  decolorize  the  ground  substance  in  which  the  bacteria 
lie. 

Under  such  circumstances,  the  cover-glasses,  after  being 
prepared  with  the  bacteria  and  allowed  to  dry,  are  to  be  left  for 
ten  minutes  in  a  capsule  or  saucer  of  20  per  cent,  solution  of 
acetic  acid  ;  after  which  they  are  washed  in  distilled  water,  al- 
lowed to  drain  and  to  dry,  edgewise,  on  filter-paper,  and  after- 
wards stained  in  the  usual  manner,  the  subsequent  washings 
being  carried  out  without  acetic  acid.  It  is  not  requisite  to  pass 
the  films  across  the  flame  at  any  stage  of  the  procedure,  even  if 
the  cover-glasses  have  been  prepared  from  broth  cultures.  Even 
this  at  times  fails  to  give  clean  preparations  (as,  e.g.,  in  the  case  of 
the  glanders  bacillus) ;  the  use  of  saturated  alcoholic  solution  of 
gentian  violet,  left  on  the  cover- glass  for  an  hour  (beneath  a 
watch  glass),  will  often  then  give  satisfactory  results  ;  the  wash- 
ing in  such  circumstances  should  be  carried  out  rapidly  in  water 
to  which  no  acetic  acid  has  been  added. 


An  Atlas  of  the  Bacteria  Pathogenic  in 
the  Human  Subject. 

PART  II. 

As  stated  in  Part  L,  the  object  of  this  Atlas  is  to  present 
graphically  from  original  preparations  the  chief  bacteria  which 
are  pathogenic  in  the  human  subject  The  figures  now  intro- 
duced comprise  nearly  all  the  remaining  bacteria  of  interest. 
Certain  are  omitted  Amongst  such  omissions  may  be  named 
the  bacillus  of  influenza,  diplococcus  intra-cellularis,  bacillus 
enteritidis,  bacillus  melitensis,  streptothrix  pseudo-tuberculosa, 
etc. ;  and  time  will  surely  add  yet  others  as  causative  agents,  es- 
pecially in  the  group  of  infective  granulomata. 

Amongst  the  micro-organisms  depicted  in  the  present  series, 
two  of  the  most  important  in  practical  medicine  are  the  typhoid* 
and  diphtheria  bacilli;  and  a  succinct  statement  may  be  made 
in  regard  to  the  bacteriological  diagnosis,  firstly  of  typhoid  fever, 
and  secondly  of  diphtheria. 

Typhoid  Fever. — Although  the  bacteriological  diagnosis 
of  typhoid  during  life  can  be  made  from  the  discovery  of  the 
typhoid  bacillus  in  the  stools  or  the  urine,  such  methods  are  of 
little  practical  avail,  necessitating,  as  they  do,  a  well- equipped 
laboratory  and  the  expenditure  of  much  time. 

Widal's  Reaction. — However  interesting  it  might  be  to  give 
an  account  of  the  observations  of  previous  investigators  which 
led  up  to  Widal's  application,  neither  space  nor  the  main  object 
of  the  present  article  will  allow  of  it.  Widal  was,  however,  the 
first  to  demonstrate  that  a  reaction,  which  had  been  previously 

*  Other  illustrations  of  this  are  given  in  Part  I. 

53 


54  PATHOGENIC    BACTERIA 

shown  to  take  place  between  the  serum  of  animals  experimen- 
tally immunized  against  certain  diseases  and  cultures  of  the  spe- 
cific bacilli  producing  such,  could  be  obtained  during  the  period 
of  infection,  and  in  this  way  serve  as  a  means  of  clinical  diagno- 
sis. If  thte  blood  serum  of  an  animal  which  has  been  rendered 
immune  against,  e.  g.,  the  typhoid  bacillus  be  added  in  a  tost 
tube  to  a  living  broth  culture  of  the  same  microbe,  the  bacilli  in 
the  culture  rapidly  cohere  and  subside  to  the  bottom  of  the  tube. 
This  is  known  as  " clumping"  or  "agglutination,"  and  is  taken 
to  indicate  the  presence  of  an  agglutinating  substance  or  "  agglu- 
tinin"  which  has  formed  in  the  blood  in  consequence  of  the 
presence  of  the  bacilli  experimentally  introduced  into  the  ani- 
mal. The  blood  serum  acquires  this  property,  however,  before 
immunization  is  established,  i.  e.,  during  the  progress  of  infec- 
tion itself,  and  the  property  may  be  utilized  as  a  means  of  diag- 
nosis, for  the  important  reason  that  the  serum  of  a  typhoid 
patient,  whilst  it  will  agglutinate  typhoid  bacilli,  will  not  agglu- 
tinate those  of  other  kinds.  What  is  true  in  the  case  of  typhoid 
is  true  also  of  other  bacterial  diseases.  The  phenomenon  admits 
of  two  applications;  a  disease  may  be  diagnosed  by  the  action  of 
the  blood  serum  upon  a  known  bacillus;  or  a  bacillus  may  be 
diagnosed  by  the  action  of  a  known  "immunized  serum."  It 
is  to  the  former  that  importance  attaches  in  practical  medicine 
and  surgery. 

After  this  brief  explanation  of  the  rationale  of  Widal's  re- 
action, the  method  of  carrying  it  out  may  be  described. 

The  broth  culture  of  typhoid  bacillus  used  must  not  be 
more  than  of  twenty-four  hours'  growth,  and  must  be  grown  in 
the  incubator  at  37°  C,  preferably  in  a  broth  that  is  not  alka- 
line, but  amphoteric  (giving  simultaneously  an  acid  and  an  alka- 


OF   THE   HUMAN   SUBJECT.  55 

line  reaction  to  blue  and  red  litmus  paper).  There  arises  even 
at  this  point  the  first  possible  source  of  fallacy.  If  such  a  broth 
culture  be  examined  by  the  hanging-drop  method  (fully  de- 
scribed in  the  previous  volume),  clumps  of  bacilli  are  not  rarely 
encountered,  and  these,  at  times,  of  considerable  size.  In  all 
cases,  then,  the  broth  culture  is  to  be  first  passed  through  a 
small  double  cone  of  filter-paper  into  a  watch-glass,  in  order  to 
remove  any  such  clumps  present;  and  after  this,  a  hanging  drop 
is  to  be  prepared  for  comparison  with  the  result  obtained  when 
the  reaction  is  tried  for.  One  important  difference  to  be  ob- 
served in  the  microscopic  preparations  is  that  although  in  the 
broth  culture  clumps  may  be  met  with,  the  field  between  con- 
tains varying  numbers  of  bacilli  in  active  movement;  whereas 
when  clumping  occurs  from  the  action  of  the  serum  of  a  typhoid 
patient,  the  bacilli  become  motionless  between  the  bacterial  isl- 
ands, if  any  remain  incoherent. 

How  to  test  the  Blood  Serum. — The  reaction  is  obtainable 
quite  early  in  the  course  of  the  disease,  as  early  as  the  fifth  day, 
and  it  persists  during  convalescence,  but  for  an  extremely  vari- 
able period  afterward.  Not  rarely  the  reaction  will  continue  for 
a  year,  but  it  may  last  many  years,  and  might  without  enquiry 
into  a  patient's  history  be  erroneously  taken  to  prove  the  exist- 
ence of  typhoid  on  the  occasion  of  some  subsequent  illness  sug- 
gestive of  it. 

The  blood  to  be  used  in  the  test  is  withdrawn  either  from, 
the  lobe  of  the  ear  or  from  the  back  of  the  finger  near  the  root 
of  the  nail;  and  the  puncture  is  best  made  by  means  of  a  surgi- 
cal or  other  neede  with  a  cutting  edge  as  well  as  a  sharp 
point. 

If  the  test  cannot  be  carried  out  at  the  time,  the  blood  must 


56  PATHOGENIC   BACTERIA 

be  collected  in  a  pipette  of  the  kind  represented  in  the  adjoin- 
ing wood  cut  (Fig.  5),  by  breaking  off  the  sealed  ends  of  the 


Fig.  5.— Pipette  containining  blood  which  has  separated  into  clot  and  serum,  the 
former  occupying  the  lower  half  of  the  bulb  (nat.  size). 

capillary  to  be  on  either  side  of  the  bulb,  and  applying  one  end 
to  the  issuing  blood.  When  the  expanded  part  is  filled,  the  ends 
are  hermetically  sealed  in  the  edge  of  the  flame  of  a  spirit-lamp. 
Blood  so  stored  can  be  tested  at  leisure,  and  (if  kept  in  the  dark) 
retains  its  qualities  for  long  periods.  Even  if  dried  the  blood 
will  provide  the  reaction;  for  this  purpose  it  is  collected  on  a 
series  of  cover-glasses,  which,  after  being  allowed  to  dry,  may 
be,  if  necessary,  posted  to  an  expert  for  examination.  If  dried 
blood  is  used,  a  solution  of  the  specific  substances  in  it  is  ob- 
tained by  means  of  distilled  water;  but  the  method  is  an  inferior 
one,  owing  to  the  difficulty  of  estimating  the  dilution  reached — 
a  matter  of  cardinal  importance. 

Nine  loop-fulls*  of  the  filtered  broth  culture  of  typhoid  are 
placed  separately  and  fairly  close  together  on  an  ordinary  micro- 
scopic slide,  the  loop  or  ose  being  introduced  as  many  times  into 
the  broth.  The  platinum  wire  is  now  sterilized  in  the  flame, 
and  with  it  a  single  loop  of  the  blood  serum  is  transferred  to  the 
slide  and  well  mixed  up  with  the  whole  of  the  nine  droplets  of 
broth  culture.  If  the  test  is  carried  out  on  the  spot,  a  few  drops 
of  blood  may  be  allowed  to  flow  from  the  lobe  of  the  ear  or  fin- 
ger, and  to  clot  in  a  small  test  tube  or  a  watch-glass;  the  serum 
so  furnished  will  be  ample. 

*  For  a  figure  of  the  "  loop,"  see  p.  26. 


OF  THE   HUMAK   SUBJECT.  57 

If  the  blood  to  be  used  has  been  stored  in  a  pipette,  the  two 
ends  of  this  are  broken  off,  and  the  contents  blown  gently  on  to 
a  slide.  A  hollow-ground  slide  having  been  prepared  with  a 
ring  of  vaseline,  and  a  clean  cover-glass  (before  commencing  the 
proceedings  just  described),  a  single  ose  of  the  admixed  broth 
and  sernm  is  placed  on  the  center  of  the  cover  glass  and  gently 
spread  out  so  as  to  cover  an  area  about  4  millimeters  in  diameter; 
the  cover  is  then  inverted,  placed  over  the  hollow  of  the  slide,  and 
gently  pressed  at  the  margin  so  as  to  render  the  enclosed  space 
quite  air  tight.  The  preparation  is  now  placed  beneath  the 
microscope  and  and  examined,  with  a  ^  homogeneous  immer- 
sion, or  a  ^  objective,  which  answers  perfectly  well  for  a  study  of 
the  result.  As  the  preparation  is  unstained,  much  of  the  light 
(if  -y^-  is  used)  must  be  cut  off  by  means  of  the  diaphragm — the 
bacilli  are  otherwise  scarcely  visible. 

If  WidaFs  reaction  ensue,  it  is  seen  in  the  movements  of  the 
microbes  becoming  sluggish  and  ultimately  ceasing,  whilst  they 
become  at  the  same  time  aggregated  into  clumps  of  the  kind 
represented  in  Fig.  1,  Plate  XVIII. 

The  time  allowed  for  the  observation  should  be  half  an 
hour.  If  no  reaction  has  ensued  within  this  time,  the  result  is 
to  be  reckoned  negative,  and  the  existence  of  typhoid  may  be 
excluded,  not  with  absolute  certainty,  but  with  very  high  proba- 
bility. In  the  case  of  such  a  negative  result,  similar  examina- 
tions must  be  repeated  during  the  course  of  'the  disease,  as  the 
reaction,  for  causes  not  known,  is  in  some  cases  delayed.  If, 
however,  the  bacilli  become  motionless,  even  without  any 
marked  clumping,  or  if  they  become  motionless,  and  clump,  the 
result  is  to  be  reckoned  as  positive. 

Before  deducing  the  existence  of  typhoid  in  these  circum- 


58  PATHOGENIC    BACTERIA 

stances,  nevertheless,  the  result  of  farther  dilutions  must  be 
tested,  for  the  reason  that  the  reaction  ensues  in  certain  cases  in 
which  typhoid  does  not  enter  into  the  question,  and,  moreover, 
that  the  blood  of  healthy  persons  possesses  at  times  an  unusual 
degree  of  the  agglutinizing  power  which  is  normally  present. 

The  diagnosis,  it  may  be  said,  becomes  strengthened  in  pro- 
portion as  the  reaction  persists  on  dilution.  In  certain  instances 
it  has  been  ascertained  that  a  dilution  of  1  to  5,000  will  yet  suf- 
fice for  its  production,  and  even  a  considerably  further  dilution 
than  this.  The  test  is  to  be  repeated,  therefore,  with  a  dilution 
of  1  in  20  (i.e.,  1  part  of  serum  to  19  of  the  broth  culture),  and 
if  still  observed,  with  a  dilution  of  1  in  50.  The  last  limit  may 
be  held  to  suffice  for  the  exclusion  of  other  possible  sources  of 
phenomenon,  and  to  establish  a  diagnosis  of  typhoid.  The  dilu- 
tion of  1  in  20  is  best  made  by  placing  nineteen  loops  of  the  broth 
separately  on  a  slide,  and  mixing  with  a  single  loop  of  the  serum; 
that  of  1  in  50,  by  diluting  1  loop  of  serum  with  10  of  distilled 
water,  and  mixing  one  loop  of  this  with  four  loops  of  the  broth 
culture. 

After  use,  all  the  slides,  cover-glasses,  and  other  materials 
are  to  be  disinfected  in  a  1  in  20  carbolic  acid  solution. 

Diphtheria. — For  the  bacteriological  investigation  of  a 
supposed  case  of  diphtheria  it  is  necessary,  firstly,  to  make  a 
microscopic  examination  of  a  culture  from  the  throat  or  nasal 
passages ;  and  secondly,  if  the  investigation  is  to  be  complete, 
to  inoculate  animals  with  a  pure  culture  in  order  to  test  the  de- 
gree of  virulence  which  the  bacillus  possesses,  i.e.,  both  the 
morphological  and  the  physiological  characters  of  the  microbe 
should  be  determined. 

The  inoculation  of  test  tubes  for  the  purpose  of  dia^  nosis 


PLATE  XV [II 


Fig.  1.—W!dal'8  Reaction. 


MEDICAL  ANNUAL,  1899. 


PLATE    XVIII. 

FIG.  i.— WIDAL'S    REACTION. 

This  fi-ure  is  given  to  show  the  bacillary  clumps  which  form  when  the  blood  serum  of  a 
p.uient  sufn_nng  from  typhoid  (ever  is  added  to  a  living  broth  cultiue  of  the  typhoid  bacillus, 
the  phenomenon  being  known  as  Widal's  reaction. 

In  the  case  of  the  preparation  figured,  i  part  of  blood  serum  was  added  to  19  ot  a  twenty- 
four  hours'  (incubated)  broth  culture.  The  blood  had  been  collected  in  a  pipette  and 
allowed  to  clot ;  a  certain  number  of  red  corpuscles  are  admixed  with  the  serum. 

The  clumping  or  agglutination  of  the  bacillus  is  readily  observable  under  i-Ctri  objective, 
though  the  clump  represented  was  drawn  under  i-i2th  oil  immersion,  and  is  magnified  about 
TOGO  times. 

The  method  of  carrying  out  the  test  for  the  diagnosis  of  typhoid  fever  is  ully  given  in 
the  text. 

FIG.  2.— BACILLUS  OF  LEPROSY. 

The  figure  represents  three  highly  vacuolated  enclothelial  cells  from  a  lymphatic  gland 
secondarily  infected  in  a  case  of  leprosy  of  the  tongue.  The  cells  occurred  along  with  others 
of  similar  character  in  irregular  groups  scattered  throughout  the  gland.  The  notable 
vacuolation  (regularly  seen  in  such  "leprous  cells")  is  possibly  due  to  an  abundant 
formation  of  digestive  fluid  secreted  by  the  cell  that  it  may  destroy  and  utilise  the  bacilli. 
Highly  vacuolated  cells  at  times  hold  extremely  few  bacilli,  possibly  as  a  result  of  such  a 
process  of  destruction.  The  bacilli  mostly  lie  in  the  septa  between  or  around  the  vacuoles, 
though  when  the  vacuole  is  not  viewed  in  strict  optical  section  they  appear  to  lie  within. 

The  leprosy  bacilli  are  slender,  straight,  or  slightly  curved  rods,  very  uniform  in  breadth, 
and  fairly  so  in  length,  and  they  closely  correspond  in  size  and  general  character  with  those 
of  tuberculosis,  as  the  latter  are  met  with,  e.g:,  in  phthisical  sputum.  They  present  a  markedly 
beaded  appearance  arising  from  protoplasmic  segmentation.  They  give,  again,  the  same 
common  staining  reaction,  in  this  resembling,  moreover,  the  bacillus  of  Mnegma  ;  i.e.,  after 
being  dyed  with  carbol  fuclisine  the  bacilli  resist  decolorisation  in  a  25  per  cent,  mixture 
of  sulphuric  acid  in  distilled  water.  The  smegrna  bacillus  is  not  infrequently  present  in  the 
urine  of  both  sexes,  and  may  be  mistaken  for  that  of  tubercle.  This  error  can  be  avoided  by 
examining  the  urine  drawn  off  by  catheter,  the  smegma  bacillus  being  in  this  way  excluded. 
And  to  select  one  of  many  differential  staining  methods,  though  dyed  with  carbol  fuchsine, 
the  colour  of  the  smegma  bacillus  is  discharged  in  a  mixture  of  20  per  cent,  nitric  acid  in 
alcohol,  whilst  that  of  the  tubercle  bacillus  is  retained.  For  the  reliable  examination  of 
urine  a  centrifuge  is  indispensable. 

The  sections  of  the  leprous  gland  were  stained  for  fifteen  minutes  (without  heat)  in  carbol 
fuchsine,  passed  through  25  per  cent,  sulphuric  acid,  washed  in  water  and  counterstained  for 
five  seconds  in  a  i  per  cent,  aqueous  solution  of  methyl  blue,  after  which  they  were  passed 
through  water,  absolute  alcohol,  oil  of  cloves,  and  mounted,  finally,  in  a  solution  of  Canada 
balsam  in  xylol.  For  the  counterstaining  of  tissue  methyl  not  methyler.e  blue,  is  to  be  used  ; 
the  latter  is  almost  entirely  removed  by  the  subsequent  immersion  of  the  section  in  alcohol. 
The  counterstaining  of  cover-glass  films  of  phthisical  sputum  is  satisfactorily  carried  out  by 
means  of  an  aqueous  solution  of  methylene  blue.  (See  the  previous  volume.) 

The  Leprosy  bacilli  are  not  in  all  cases  located  within  the  cells  of  a  tissue  :  they  may  lie  in 
a  ground  substance  or  gloea  occupying  the  lymph  spaces. 


PLATE  XIX 


f/g.  r.—  Diphtheria  Bacillus  (typical  form}. 


* 


\ 


'"'...       ...  <. 

• "  -;  '- 

2.— Diphtheria  Bacillus  (atypical). 


Fig.  8.— Diphtheria  Bacillus  (atypical).  Fig.  4.— Hoffmann's  Pseudo-Diphtheria  Bacillus. 


MEDICAL  ANNUAL,   1899. 


PLATE    XIX. 

BACILLUS     OF     DIPHTHERIA. 

FIG.  i.— THE  "TYPICAL,1    "  KLEBS-LoFFLER,"   OR   "LONG"   DIPHTHERIA 

BACILLUS. 

Fro  n  a  culture  on  Loffler's  blood-serum  ot  twenty-two  hours'  age,  incubated  at  37°  C.  The 
growth  was  the  third  sub-culture  of  the  original  from  the  throat.  Stained  with  Lijffler's 
methylene  blue,  washed  in  tap  water.  The  case  from  which  the  culture  was  raised  concerned 
a  boy  (P.  R.J,  admitted  to  St.  Thomas's  Hospital,  October  iSih,  1897,  with  inflamed  throat ; 
there  was  much  membrane  on  the  tonsils,  and  in  the  larynx  as  evidenced  by  the  stridor  and 
retraction  of  the  chest.  Tracheotomy  was  performed,  the  tube  being  removed  on  the  founh 
day.  Four  thousand  units  of  diphtheria  antitoxin  were  administered  by  subcutaneous 
injection.  Until  the  early  part  of  November  progress  had  been  favourable  ;  on  November  24 th 
palatal  paralysis  was  noted,  the  voice  acquiring  a  nasal  twang;  this  slowly  improved.  The 
knee-jerks  were  absent  on  November  agth,  and  still  absent  on  December  24th,  but  the  patient 
could  at  that  date  swallow  without  regurgitation. 

The  Bacilli  are  straight  or  slightly  curved  rods  of  varying  length  and  thickness,  often  set  in 
parallel  groups  of  two  or  more.  The  designation  of  "long"  implies  that  many,  not  all,  are  of 
conspicuous  length.  One  end  (or  both)  may  be  enlarged  or  bulbous  ;  in  the  absence  of  this, 
the  ends  are  abruptly  rounded  without  tapering.  The  bacilli  present  a  notable  segmentation 
of  protoplasm,  which  is  divided  into  deeply  stained  parts ;  these,  which  may  be  far  from 
equidistant,  are  in  some  instances  flattened  across  the  long  axis  of  the  rod,  in  others,  spherical. 
The  extremities  of  the  bacillus  correspond  with  terminal  segments.  In -some  of  the  shorter 
rods  only  terminal  parts  are  dyed — end  or  polar  staining. 

The  primary  forms  appear  as  short  uniformly  stained  bacilli,  with  ends  slightly  smaller  than 
the  rest  of  the  rod  ;  these  undergo  transverse  division,  before  the  completion  of  which  they 
appear  as  diplo-bacilli  with  the  opposed  ends  flattened.  Segmentation  rapidly  takes  place, 
the  shortest  forms  exhibiting  onh  end-staining ;  the  hitter  are  distinguishable  from  true 
diplo-bacilli  by  the  absence  of  tapering  free  ends  and  the  length  of  unstained  centre.  These 
forms  of  the  diphtheria  bacillus  are  classed  as  "  typical,"  since  they  are  commonly  associated 
with  the  typical,  more  virulent  examples  of  diphtheria;  yet  not  invariably  so,  for  not  only 
may  ''atypical"  forms  be  highly  virulent,  but  similar  "typical"  or  "long"  forms  may  possess 
little  palhogenicity  (as  tested  upon  the  guinea-pig),  and  occur  in  cases  which  clinically  present 
no  other  features  than  those  of  sore  throat,  unaccompanied  even  with  any  marked  malaise. 

Fu;.  2.— "ATYPICAL"  OR  "SHORT"  DIPHTHERIA  BACILLI. 
A  culture  of  thirteen  hours'  growth  on  Loffler's  blood  serum,  carried  on  !rom  one  (Viennese) 
of  the  virulent  strains  in  use  at  the  Conjoint  Laboratories  of  the  Royal  College  of  Physicians 
and  Surgeons,  London,  for  the  preparation  of  toxin  employed  in  the  production  of  diphtheria 
antitoxin  from  the  horse.  Besides  a  few  segmented  forms  are  others  of  an  "atypical "  kind, 
commonly  ranged  in  parallel  collections  of  two  or  more  ;  these  mostly  taper  off  at  the 
extremities,  presenting  a  deeply  stained  centre,  which  is  usually  divided  by  an  uncoloured 
narrow  line  indicative  of  an  incompleted  transverse  fission,  the  elements  being  double,  or 
diplo-bacilli.  Beyond  the  deeply  stained  centre  the  bacillus  is  of  a  lighter  blue.  Here  and 
there  a  pyrifonn  element  occurs,  more  deeply  stained  towards  the  larger  end  ;  or  short  deep'y 
stained  spindles. 

FIG.    S.-A   SECOND    EXAMPLE    OF   THE    "ATYPICAL"    OR    "SHORT" 
DIPHTHERIA    BACILLUS. 

From  a  virulent  case  of  the  disease.  Diplo-bacilli  in  parallel  groups  ;  beyond  their  opposed 
deeply  stained  central  ends  the  component  elements  taper  off  and  are  but  lightly  coloured. 
Pear-shaped  forms  are  present,  and  a  few  which  exhibit  polar  and  segmented  staining.  From 
its  staining  reaction  the  form  has  been  named  the  "sheath"  variety  by  Dr.  J.  Eyre,  to  whom 
the  author  is  indebted  for  the  preparation  from  which  the  figure  has  been  drawn.  The  "  sheath/' 
variety  occurs,  as  a  rule,  in  the  milder  types  of  diphtheria.  It  is  but  rarely  met  with,  and  is 
not  strictly  stable  ;  if  grown  for  some  days  upon  alkaline  potato  and  again  sown  on  blood- 
scrum,  it  acquires  the  segmented  characters  of  the  "typical"  form. 

Another  atypical  pathogenic  variant  has  been  described  of  the  same  form  as  the  above,  but 
staining  uniformly  and  deeply  throughout. 

FIG.  4.— HOFMANN'S    BACILLUS. 

Culture  of  twenty-four  hours'  on  agar,  incubated  at  37°  C.  Stained  with  Loffler's  blue, 
washed  in  tap  water.  The  culture  was  carried  on  from  one  isolated  by  Dr.  E.  A.  Peters, 
to  whom  the  author  is  indebted  for  a  sample  of  the  strain.  Diplo-bacilli,  occurring  in 
parallel  groups  of  two  or  more.  The  elements  composing  a  single  diplo-bacillus  are  short, 
squat,  wedge-shaped,  with  opposed  bases,  and  stain  uniformly  throughout.  They  are 
markedly  shorter  than  the  atypical  or  short  varieties  of  the  diphtheria  bacillus,  and  relatively 
broader  at  their  bases.  In  older  cultures  segmented  and  irregular  involution  forms  may  be 
encountered.  Hofmann's  bacillus  (sometimes  named  a  pseudo-diphtheria  bacillus)  is  not 
found  as  a  cause  of  true  diphtheria  in  the  human  subject.  Hence,  though  isolated  in  many 
forms  of  sore  throat,  such  lesions  are  not  to  be  regarded  as  diphtheritic.  The  bacillus,  may, 
however,  be  associated  with  the  "  typical,"  "  Klebs-Loffler,"  or  "  long"  diphtheria  bacilli  in 
diphtheritic  affections,  but  under  such  circumstances  its  presence  may  be  regarded  as  of 
secondary  significance. 


PLATE  XX 


- 


1.—  Proteus  Vulgarts., 


Fig.  2.— Proteus  Vulgar!*  (the  early 
filamentous  phase). 


MEDICAL  ANNUAL,  i8gg. 


PLATE    XX. 

BACILLUS    PROTEUS    VULGARIS. 

This  organism  is  introduced  as  one  of  the  most  common  of  those  causing  putrefaction, 
though  different  varieties  of  the  bacillus  coli  are  almost  as  ubiquitous. 

Although  by  some,  putrefactive  organisms  are  not  classed  as  pathogenic,  because  they  are 
not  the  causes  of  any  process  differentiated  clinically  as  a  disease,  they  are  pathogenic  in 
the  wider  and  truer  sense,  since  putrefaction  plays  so  important  a  part  in  the  sepsis  of 
wounds  and  in  the  toxa;mia  accompanying  cancrum  oris,  the  ulceration  of  extensive  carcino- 
mata  of  the  alimentary  and  respiratory  passages  and  conditions  of  a  like  kind. 

FIG  2. 

An  ''  Impression  preparation1'  from  a  gelatin  culture  of  eighteen  hours. 

Carbol  fuchsine,  washed  in  water  weakly  acidulated  with  acetic  acid.  The  culture  was 
made  by  streaking  a  Petri  capsule  (after  the  jelly  poured  into  it  had  set)  with  a  straight 
platinum  wire  infected  from  a  pure  culture  of  the  organism.  The  impression  preparation  is 
obtained  by  allowing  a  cover-glass  to  fall  upon  some  part  of  the  streak  of  growth  and  then 
gently  raising  it  by  one  edge,  when  the  line  of  culture  is  brought  away  adhering  to  the  under 
side  of  the  glass  ;  the  specimen  is  then  stained  in  the  usual  way. 

The  preparation  shows  the  early  or  filamentous  stage  in  the  growth  of  the  bacillus,  which 
is  highly  pleomorphic,  whence  its  name  of  Proteus. 

The  streak  (of  which  one  edge  is  depicted)  consists  of  long,  closely-matted,  unbranched 
filaments  arranged  in  strands.  As  seen  at  the  free  margin,  some  are  sharply  re-curved  upon 
themselves. 

The  organism  figured  was  isolated  from  macerating  muscle,  the  actual  material  being  beef 
steak,  which  was  minced  and  incubated  in  distilled  water  at  37°  C. 

FIG.  i. 

Ail  impression  preparation  of  a  similar  streak  culture  at  a  later  stage  when  the  gelatin 
was  in  process  of  liquefaction.  Carbol  fuchsine,  washed  in  acidulated  water. 

The  filaments  have  mostly  divided  into  short  rods,  often  constricted  across  the  middle  or 
in  pairs  end-to-end  as  diplo-bacilli.  Here  and  there  longer  rods  occur  and  filamentous 
forms,  but  the  longer  of  the  latter  have  not  been  introduced. 


PLATE  XXI 


-\' 


I 

**      N. 


U 


o<  ' 


•:     i 

Fig.  1.— Bacillus  of  Glanders. 


.  2.— Bacillus  Tuberculosis,  in  a  pure 
culture. 


MEDICAL  ANNUAL,  1899. 


PLATE    XXI. 

FIG.  i. -BACILLUS    OF    GLANDERS    (BACILLUS    MALLEIJ. 

From  the  growing  edge  of  a  culture  on  glycerin-agar  of  three  days'  growth,  incubated 
at  37°C. 

Carbol  fuchsine,  washed  in  diluted  acetic  acid.  The  culture  was  made  from  a  potato  growth 
of  characteristic  honey-yellow  colour,  raised  from  a  glandered  horse,  the  potato  culture  being 
the  second  remove  only  from  the  original. 

The  bacillus  (which  is  very  difficult  to  stain  with  certainty  in  the  tissues)  may  be  stained 
in  cover-glass  films  with  aqueous  solution  of  gentian  violet  (ten  minutes),  followed  by  washing 
in  i  in  10,000  caustic  potash  solution  and  afterwards  tap  water  ;  or,  by  means  ot  alcoholic 
solution  of  gentian  violet  for  one  hour,  rapidly  washed  in  water.  The  action  of  Loffler's 
blue,  for  cover-glass  films  is  uncertain. 

Somewhat  slender  rods  of  varying  length  and  thickness,  straight  or  slightly  curved,  the 
shortest  appearing  as  ovals,  the  longer  as  unsegmented  filaments  which  are  commonly  less 
deeply  stained  ;  some  of  the  filaments  present  were  longer  than  those  figured.  Here  and 
there  end-to-end  pairs  are  met  with.  In  some  of  the  rods  one  or  more  minute,  sharply- 
defined,  circular  vacuoles  are  present.  A  few  of  the  bacilli  present  a  inoniliforin  outline,  but 
none  any  distinct  segmentation  of  protoplasm  or  "  beading,"  as  they  may  in  the  tissues. 

The  organisms  tend  to  cohere  in  clusters,  some  of  the  smaller  of  which  are  selected  in  the 
illustration. 

FIG.  c.-TUBERCLE    BACILLUS. 

Pure  culture  on  Luffler's  blood-serum,  incubated  at  37°  C.,  carried  on  from  a  growth  on 
glycerin-agar  which  was  raised  from  the  lymphatic  gland  of  a  guinea  pig,  inoculated  from  a 
case  of  tubercular  pleurisy  in  the  human  subject. 

The  growth  on  the  serum  progressed  slowly  and  took  the  form  of  a  thin,  white  film  in 
which  were  sparsely  scattered,  thicker,  more  opaque  areas. 

Stained  with  ca.'bol  fuchsine  warmed  on  the  cover-glass,  and  treated  with  25  per  cent, 
sulphuric  acid  in  a  watch-glass.  If  used  in  the  cold,  the  dye  should  be  allowed  fifteen 
minutes. 

The  culture  consists  of  straight  and  slightly-curved  rods  with  rounded  ends,  and  of  varying 
length,  the  shortest  hardly  more  than  oval.  The  bacilli  tend  to  occur  in  small  parallel 
groups  arranged  in  irregular  lines,  or  set  at  .various  angles  to  one  another  in  away  suggestive 
of  Chinese  characters.  They  exhibit  none  of  the  segmentation,  or  "  beading,"  so  commonly 
presented  in  phthisical  sputum  (see  Plate  XXX  in  the  previous  volume). 

Branching  Forms. — On  the  right  are  shown,  from  the  same  culture,  two  examples  of  the 
filamentous  forms,  which  recall  the  branched  mycelium  of  the  hyphal-fungi  or  moulds. 

The  colouration  of  the  bacilli  (stained  by  Gram's  method)  is  almost  limited  to  the  minute 
spherical  granules  lying  within  the  bacillary  cell._  _ 

Branching  forms  are  at  times  met  with  in  phthisical  sputum. 


PLATE    XXII. 

FIG.  i. -BACILLUS    OF    RHINOSCLEROMA. 

From  a  streak  culture  on  agar,  of  forty-eight  hours'  growth,  incubated  at  37°  C. 

Carbol  fuchsine,  washed  in  dilute  acetic  acid.  The  figure  shows  a  group  of  bacilli  united 
into  a  zooglcea  by  an  abundant  ground  substance  which  is  faintly  stained  with  the  dye. 
The  micro-organism  consists  of  spherical  elements  occurring 'singly,  but  most  frequently  in 
pairs  or  short  chains.  Rod  forms  may  also  be  met  with. 

As  showing  the  essential  similarity  of  ground-substance,  or  gloea,  and,  that  which  con- 
stitutes a  bacterial  capsule,  it  will  be  noticed  that  whilst  at  the  periphery,  where  active 
multiplication  is  proceeding,  the  bacilli  lie  closely  embedded  in  this  substance,  more 
centrally,  where  the  latter  has  accumulated  in  larger  amount,  it  has  parted  into  areas 
appertaining  to  definite  groups,  showing  the  divided  share  which  the  organisms  have  taken 
in  its  production.  At  the  lower  end  of  the  figure,  near  its  middle,  there  is  a  diplo-bacillus 
quite  isolated  from  the  neighbouring  mass,  and  furnished  with  a  capsule  proper  to  itself. 

FIG.  2.— STREPTOTHRIX    ACTINOMYCES. 

Broth  culture,  of  seven  days'  age.  incubated  at  37°  C. 

In  broth  the  growth  occurs  as  small  spherical  colonies,  which  are  best  examined  by  being 
teased  out  on  a  cover-glass  after  haying  been  washed  in  distilled  water.  The  preparation  is 
allowed  to  dry  and  may  be  then  stained  with  carbol  fuchsine  or  aqueous  solution  of  gentian 
violet ;  in  the  former  case  the  subsequent  washing  is  carried  out  with  acidulated  water,  in  the 
latter  with  tap  water,  or.  what  is  better,  with  a  i  in  10,000  solution  of  caustic  potash, 
followed  by  tap  water. 

The  culture  consists  solely  of  long,  interlacing,  slightly  wavy  filaments  which  give  off 
lateral  shoots.  The  branches  vary  in  length  according  to  their  age,  taking,  on  their  first 
appearance,  the  form  of  minute  excrescences. 

Cultures  of  the  streptothrix  Madura?  (the  cause  of  mycetoma  or  Madura  disease)  are  indis- 
tinguishable in  microscopic  features  from  streptothrix  actinomyces. 

As  found  in  the  tissues  of  oxen  affected  with  actinomycosis,  the  filaments,  as  a  rule, 
terminate  in  clubbed  enlargements,  and  radiate  from  a  central  mass.  In  the  lesions  of  the 
human  subject  the  clubs  are  by  no  means  regularly  present,  nor  is  a  radial  disposition  of  the 
filaments  always  obvious. 

Such  differences  possibly  indicate  that  the  streptothrix  causing  the  disease  known  clinically 
as  actinomycosis  in  man  and  the  lower  animals  is  not  always  identical,  the  existence  of 
pathogenic  varieties  (which  have  received  special  names)  having  been  demonstrated  by 
means  of  artificial  cultures. 

Owing  to  its  anomalies,  the  classification  of  this  group  of  organisms  has  been  long  a 
subject  of  controversy,  which  has  been  terminated,  at  least  for  a  while,  by  raising  it  into  a 
distinct  class  of  fungi  under  the  name  of  streptothricise. 

The  group  includes  non-pathogenic  as  well  as  pathogenic  forms,  and  comprises  micro- 
organisms which,  like  hyphal-fungi  or  moulds,  form  a  mycelium  of  branching  filaments 
originating  from  spherical  spores  ;  certain  of  the  filaments  (like  the  aerial  hyphse  of  moulds) 
ru>e  into  the  air  from  the  mycelium  and  produce  at  their  extremities  chains  of  spherical 
elements  comparable  to  spores,  though  of  a  different  morphological  nature  from,  e.t;,,  the 
endo-spores  of  bacilli. 


PLATE  XXIL 

>?••->••'..,. 


^w 


../:  ;;  /"  ;' 

;:  ..  ,      -  >  J     -^ 


Fig.  2.—Streptothrix  Actinomyces. 

MEDICAL  ANNUAL, 


PLATE    XXIII. 

FIG.  i. -STREPTOCOCCUS    PYOGENES. 

Broth  culture  of  forty-eight  hours'  growth,  incubated  at37°C.,  from  the  pus  of  one  of  the 
subcutaneous  abscesses  which  arose  during  the  course  of  puerperal  septicaemia. 

Carbol  fuchsine,  washed  in  dilute  acetic  acid.  The  chain  on  the  right  hand  side  of  the 
figure  is  added  from  a  cover-^lass  preparation  of  the  pus  of  an  axillary  abscess  which  showed 
large  numbers  of  such  without  any  staphylococci.  The  micro-organism,  which  is  best 
studied  in  broth  cultures,  presents  itself  as  micrococci  arranged  in  chains  of  varying  length. 
The  component  elements  are  almost  everywhere  Hattened,  and  occur  in  pairs,  showing  that 
active  division  is  in  progress  throughout  the  chain.  Here  and  there  elements  occur  which 
are  oval  or  elongated  in  the  direction  of  the  chain,  and  all  transitions  may  be  traced  between 
such  and  the  pairs  of  flattened  cocci  resulting  from  their  sub-division.  Wedge-shaped 
forms  may  be  met  with  in  adaptation  to  the  pressure  arising  at  sudden  bends.  At  times 
division  of  a  component  co  cus  takes  place  in  the  long  axis  of  the  chain,  as  may  be  seen  in 
that  on  the  right  hand  side.  This  mode  of  fission  may  become  a  source  of  lateral  branching 
should  the  process  of  sub-division  continue  in  parallel  planes. 

At  times  certain  of  the  cocci  will  divide  cross-wise  into  four.  Some  of  the  chains  are 
very  slender,  and  different  parts  of  the  same  chain  may  present  marked  differences  in  respect 
of  breadth  or  thickness. 

FIG.  2.— BACILLUS    OF   QUARTER-MVIL,   OR    SYMPTOMATIC    ANTHRAX. 

Cultivation  in  2  per  cent,  glucose  broth,  incubated  at  37°  C.,  of  twenty-four  hours'  growth. 

The  organism,  like  those  of  malignant  oedema  and  tetanus,  is  a  strict  anaerobe,  i.e.,  it 
grows  only  in  the  absence  of  gaseous  oxygen.  The  culture  was  grown  in  an  atmosphere  of 
nitrogen  by  Buchner's  method  of  removing  the  atmospheric  oxygen  with  pyrogallate  of 
potassium.  Carbol  fuchsine,  washed  in  water  weakly  acidulated  with  acetic  acid. 

The  growth  at  this  stage  consists  of  rods  of  varying  length  produced  either  into  simple 
or  segmented  filaments.  The  elements  composing  the  filaments  are  by  no  means  of  regular 
length,  in  consequence  of  a  continuance  of  their  sub-division. 

The  breadth  is  less  than  that  of  the  anthrax  bacillus,  and  the  apposed  ends  of  the  segments, 
in  place  of  being  squarely  cut  (as  in  the  latter  ;  see  preceding  volume),  are  rounded. 

It  must  be  here  observed,  however,  that  in  the  bacillus  of  anthrax  squareness  of  the 
apposed  ends  may  be  as  wanting  as  in  either  malignant  oedema  or  quarter-evil. 

At  the  end  of  twenty-four  hours  few  sporulating  rods  were  present  in  the  culture. 

By  the  third  day  abund  ;nt  spore-formation  had  taken  place,  as  is  shown  in  Fig.  3.  The 
preparation  consists  of  straight  rods,  large  numbers  of  which  are  sporing.  Pairs  of  rods 
joined  end-to-end  are  not  infrequent.  The  spore  forms,  as  a  rule,  at  one  of  the  extremities 
of  the  rod,  which  it  considerably  exceeds  in  diameter  so  as  to  give  rise  to  a  drum-stick 
appearance,  much  as  in  the  tetanus  bacillus,  except  that  the  spores  are  oval  in  place  of 
being  spherical. 

The  formation  of  the  spore  is  first  evidenced  by  a  swelling  of  the  end  of  the  rod  :  in  this 
enlargement  the  unstained  spore  subsequently  appears. 

This  organism  is  closely  like  that  of  malignant  cedema  both  in  its  cultural  characters  and 
its.  morphology.  In  the  bacillus  of  malignant  cedema,  however,  the  spores,  in  place  of 
bsinsr  terminal,  form  towards  the  centre  of  the  rods.  The  bacillus  of  quarter-evil  has  not 
yet  been  identified  as  a  cause  of  disease  in  man  ;  that  of  malignant  redema  not  infrequently 
has. 

Stain  :  Aqueous  solution  of  gentian  violet,  washed  in  i  in  10,000  solution  of  caustic  potash, 
followed  by  tap- water. 


PLATE  XXIII 


?*..••. 


'*""" 


„• : 
*  •  v 


«~  * 


»* 
'•-* 


«••.!•„••'." 
.»t"  > "     » 


/   r 


F/g.  1.— Streptococcus  Pyogenea. 


Fig.  2.~Bocitlu8  of  Quarter  Evil. 


/' 


.  3.—  Bacillus  of  Quarter  Euil 
(sparing  stage). 


MEDICAL  ANNUAL, 


PLATE    XXIV 

FIG.  i.— DIPLOCOCCUS    PXEUMONL-E. 

Culture  on  Loffler's  blood-scrum,  of  twenty-four  hours'  age,  incubated  at  37°  C. 

Stained  by  Gram's  method.  The  cover-glares  having  been  prepared  in  the  usual  way, 
are  immersed  ina  watch-glass  of  aniline  gentian  violet  for  ten  minutes,  passed  through  water, 
and  then  placed  in  Gram's  iodine  solution  for  five  minutes,  after  which  they  are  washed  in 
alcohol  until  no  further  colour  comes  away  ;  they  are  then  placed  on  edge  to  dry,  and  finally 
mounted  in  xylol  balsam. 

The  cocci  occur  singly  and  in  pairs,  but  mostly  grouped  in  rows  of  varying  length.  In  the 
particular  strain  shown  (which  was  isolated  by  Dr.  J.  W.  Wash  bourn  and  Dr.  J.  Eyre, 
and  found  highly  virulent  when  tested  upon  animals'  the  chain  formation  is  unusually  pro- 
nounced, the  organism  being  a  streptococcal  variant. 

Considerably  longer  chains  than  the  longest  depicted  were  present.  The  cocci  composing 
the  chains  are  actively  subdividing,  as  evidenced  by  the  flattened  pairs  of  which  they  so 
largely  consist.  Here  and  there  unusually  large  elements  occur  in  which  sub-division  has 
not  yet  taken  place. 

In  pure  cultures  the  organism  is  unprovided  with  the  capsule  which  it  presents  when 
studied  in  the  sputum  and  pulmonary  tissue  in  cases  of  acute  pneumonia. 

FIG.  2. 

A  preparation  of  the  heart-blood  of  a  rabbit  expejimentally  infected  with  the  foregoing 
strain  of  diplococcus  pneumoniae  by  intra-peritoneal  injection.  All  the  organisms,  whether 
single  cocci  or  pairs,  are  surrounded  with  a  thick  capsule.  The  specimen  was  dyed  with 
the  following  modification  of  dahlia  stain  devised  by  Dr.  A.  MacConkey :  Dahlia,  '5  grms. ; 
methyl  green  (oo  crystal),  1*5  grms.;  saturated  alcoholic  solution  of  fuchsine,  10  c.cm.  ; 
distilled  water  to  200  c.cm. 

The  dahlia  and  methyl  green  are  rubbed  up  in  a  mortar  with  part  of  the  water  until 
dissolved,  the  fuchsine  is  then  added,  and,  finally,  the  rest  of  the  water.  The  cover-glass, 
after  the  dye  is  placed  upon  it,  is  held  over  a  low  flame  until  the  steam  rises,  placed  asicl- 
for  five  minutes,  washed,  allowed  to  dry,  and,  finally,  mounted  in  xylol  balsam. 

The  appearance  of  the  capsule  under  the  conditions  of  the  experiment  and  in  the  human 
tissues  possibly  marks  a  defensive  formation  on  the  part  of  the  bacterium  to  protect  it  against 
the  action  of  the  cells  and  body  fluids  (Louis  Jenner). 

FIG.  3.-TETANUS    BACILLUS. 

Culture  in  glucose  broth,  grown  anaerobically  at  37°  C.  ;  stained  with  gentian  violet. 

Slender  rods  and  simple  filaments.  Most  of  the  rods  have  sporulated,  the  spore  being  of 
spherical  form  and  situated  at  one  end  of  the  rod  which,  in  consequence,  acquires  the 
appearance  of  a  drum-stick.  Branching  filaments  may  be  met  with,  as  in  the  case  of  the 
tubercle  bacillus. 


PLATE  XXIV 


•••s 

»       i    y 
..     '•* 


••  -v  % 

t         ••*.     .. 


•  \ ,.«--%  "•  ;  v 
"•', '•,"  X"-  /-""" 


*»••      *    .       :     « 
*   .•  / 

%  -' 


Fig,  1.—Diplococcus  Pneumonice  (pure  culture, 
encapsulated). 


%  4^i        A 
1  I  •     * 


.  2.—Diplococous  Pneumonia  (capsulated 
condition). 


.  3.— Bacillus  of  Tetanus. 

MEDICAL  ANNUAL,  2899. 


PLATE    XXV. 

FIG.  i.— BACILLUS    PNEUMONI/E    (FRIEDLANDER). 

From  a  streak  culture  i>n  agar,  of  twenty- four  ho\\rs'  growth,  incubated  at  37°C. 

Stained  wii  h  aqueous  solution  of  gentian  violet,  washed  in  caustic  potash  solution  i  part 
to  '0,000),  followed  by  tap-water. 

Thick  rods  of  varying  length,  the  shortest  appearing  as  ovals  or  even  as  coccus  fornih  or 
spheres.  Fairs  of  oval  (or  spherical)  elements,  end-to-end  nre  not  uncommon.  In  some  <•( 
the  shelter  form-;  one  or  more  circular  vacuoles  are  present. 

In  pure  cultures  the  micro-organism  has  no  capsule  such  as  it  presents  when  found  in  the 
sputum  or  lung  in  acute  pneumonia 

It  is  not  stained  by  Grain's  method,  and  in  this  contrasts  with  the  diplococcus  pneumoni.c 
which  is  found  in  othi-r  cises  of  acute  pneumonia,  in  the  sputum  and  pulmonary  tissue,  etc. 

FIG.  2. 
SPIRILLUM    OF    ASIATIC    CHOLERA    (KOCH'S    "COMMA    BACILLUS "V 

From  a  streak  culture  on  agar,  of  twenty-four  hours'  growth,  incubated  at  37° C. 

Aqueous  solution  of  gentian  violet,  washed  in  i  in  10,000  caustic  potash  solution,  followed 
t>y  tap-water. 

A  group  of  typical,  slighty-cur\ed  rods  is  shown  at  the  lowest  part  of  the  figure  ',  others 
are  selected  to  show  varieties  of  form.  The  rods  vary  in  length  as  well  as  in  thickness. 
Some  are  sharply  curved  like  the  letter  C  ;  others  are  curved  in  opposite  directions  and 
present  two  bends  like  a  shallow  S. 

Some  of  the  bacilli  exhibit  one  or  two  circular  vacuoles  ;  the  vacuole  may  occupy  one  end 
of  the  rod,  which  may  be  slightly  enlarged.  Although  commonly  rounded,  the  extremities  of 
the  bacilli  may  be  bluntly  pointed.  In  addition  to  the  simple,  curved  rods,  there  are  longer 
filamentous,  undulatory  forms  of  different  lengths;  these  present  no  traces  of  a  segmented 
or  composite  structure.  The  proper  spirillar  or  twisted  feature  of  the  filamentous  forms  is 
appreciable  only  in  a  hanging  drop  made,  e.g.,  from  a  broth  culture.  In  such  a  drop  even 
the  longest  forms  are  twisted,  as  appears  by  the  alternation  of  parts  within  and  out  of  focu«. 
In  'iried  preparations  the  curves  are  reduced  to  a  single  plane,  the  filament  becoming 
undulatory  or  serpentine  without  spiral  twist. 


PLATE  XXV 


/-.£  * 


5 


'     & 


\    * 
'•\ 


->> 


Fig.  l.—Baeillus  Pneumonia.—  FRIEDLANDER. 


Fig.  2.— Spirillum  of  Asiatic  Cholera  ("Comma 
Bacillus  "). 


MEDICAL  ANNUAL,  1899. 


OF  THE   HUMAN   SUBJECT.  77 

is  carried  out  as  follows:  The  best  culture  medium  is  Lbffler's 
blood  serum,  though  ascitic  or  pleuritic,  or  even  hydrocele  fluid 
answers  sufficiently  well ;  the  medium  is  solidified  by  heat  in 
test  tubes  placed  on  the  slant  to  furnish  a  surface  on  which  the 
microbes  may  be  sown.  The  serum  slant  may  be  inoculated  by 
means  of  the  platinum  ose  ;  this  having  been  first  sterilized  in 
the  flame  is  drawn  over  the  tonsil  or  pharynx  (over  the  mem- 
branous exudation  if  such  is  present)  and  is  then  rubbed  over 
the  whole  surface  of  the  medium  so  as  to  distribute  the  organ- 
isms present  as  widely  as  possible.  Or  the  serum  may  be  more 
effectively  inoculated  by  means  of  a  small  swab  of  cotton. 
The  swabs  for  this  purpose  are  made  by  passing  a  strip  of  cotton 
through  an  eye  at  the  end  of  a  piece  of  stout  copper  wire 
around  which  the  material  is  then  twisted  for  a  short  distance 


c 


Fig.  6.— A  reduced  figure  of  the  swab,  etc.,  described  in  the  text. 

(Fig.  6).  The  opposite  end  of  the  wire  is  bent  to  afford  a  hold, 
and  a  second  piece  of  cotton  is  wrapped  around  near  this  as  a  plug 
which  closely  fits  the  mouth  of  the  test  tube,  into  which  the 
swab  is  inserted. 

A  set  of  such  tubes  may  be  sterilized  by  being  heated  for  an 
hour  in  the  hot-air  sterilizer  at  150°  0,  and  can  be  kept  ready 
for  use.  When  about  to  be  employed  the  swab  is  removed  from 
the  tube  and  applied  to  the  most  promising  area  of  the  throat ; 
it  is  then  introduced  into  the  culture  tube  and  gently  moved 
over  the  surface  of  the  serum  The  swab  may  then  be  destroyed 


78  PATHOGENIC   BACTERIA 

in  the  flame.  After  inoculation  the  culture  tube  is  incubated 
at  37°  C,  having  been  first  capped  to  prevent  any  drying  of  the 
medium.  Incubation  of  the  tube  is  essential,  and  without  an  in- 
cubator the  further  investigation  is  preferably  handed  over  to  an 
expert  or  to  one  of  the  Associations  or  Institutes  where  such 
work  is  carried  out. 

Culture  tubes  and  swabs  may  be  obtained,  moreover,  from 
many  such  sources.  After  twenty-four  hours'  incubation  the 
growth  of  the  diphtheria  bacillus  appears  as  small  hemispherical, 
greyish,  shining  colonies;  where  such  have  arisen  so  closely  as  to 
coalesce  the  circular  outline  is  wanting.  As  a  rule,  colonies  of 
other  bacteria  develop  concurrently,  such,  e.  g.,  as  staphylococcns 
pyogenes  aureus,  staphylococcus  pyogenes  albus,  streptococcus 
pyogenes,  sarcina  lutea,  or  forms  of  yeasts.  The  colonies  of  the 
diphtheria  bacillus  cannot  be  distinguished  with  any  certainty 
by  their  macroscopic  characters,  though  yellow  colonies  may  be 
ignored.  If  no  suspicious  colonies  appear  for  individual  exam- 
ination, the  6se  is  swept  over  the  culture  and  the  material  trans- 
ferred to  cover-glasses,  on  each  of  which  a  drop  of  distilled  water 
has  been  previously  placed,  the  examination  being  made  in  the 
dried  state,  by  the  technique  fully  described  in  Part  I. 

Labor  may  be  saved  by  using  a  single,  long  cover-glass  on 
which  a  series  of  such  preparations  may  be  made  and  stained,  in 
place  of  separate  circles  or  squares. 

The  films  having  been  allowed  to  dry,  they  may  be  stained 
with  carbol  fuchsine  or  by  Gram's  method,  but  as  satisfactorily 
and  simply  with  Loffler's  methylene  blue  as  with  any  other 
dye: 

Concentrated  Alcoholic  solution  I  Solution  of  Potash  (1-10,000) 

of  Methylene  Blue        30  parts  |  100  parts 

The  stain  is  allowed  to  act  for  five  minutes  and  washed  off 


OP  THE   HUMAN"   SUBJECT.  79 

in  tap  water,  the  cover-glasses  being  then  placed  on  edge  to  dry 
or  gently  pressed  between  filter  paper;  when  quite  dry  they  are 
mounted  in  xylol  balsam. 

The  diphtheria  bacillns  is  depicted  and  described  on  Plate 
XIX. 

If  no  diphtheria  bacilli  are  found,  a  second  series  of  prepa- 
rations should  be  made  from  another  6se  of  the  same  culture, 
and  if  a  negative  result  is  again  obtained,  a  third  set.  Where 
any  doubt  enters  into  the  morphological  diagnosis  it  is  impera- 
tive to  test  the  virulence  of  the  bacillus  upon  animals.  With 
this  object  a  tube  of  broth  is  inoculated  from  a  colony  of  the  ba- 
cillus, and  after  forty  hours'  incubation  2  cubic  centimeters  of 
the  shaken  culture  are  injected  into  the  subcutaneous  tissue  of 
the  anterior  or  lateral  abdominal  wall  of  a  guinea  pig  weighing 
500  grams.  The  broth  used  for  the  culture  is  prepared  from 
minced  veal  which  is  allowed  to  ferment  in  order  to  remove  the 
glucose  present  in  it,  and  by  so  doing  to  reduce  the  amount  of 
acid  formed  by  the  bacillus,  the  production  of  which  acts  dele- 
teriously  upon  the  micro-organism  and  diminishes  the  amount  of 
toxin  elaborated  by  it. 

It  must  be  borne  in  mind  that  diphtheria  bacilli  present  all 
grades  of  virulence,  the  virulence  at  one  end  of  the  scale  dimin- 
ishing until  it  entirely  vanishes. 

There  are,  that  is  to  say,  diphtheria  bacilli  of  typical  mor- 
phological form  which  have,  presumably,  lost  their  virulence, 
and  the  injection  of  a  broth  culture  of  which  produces  neither 
local  nor  general  results  in  the  animal  upon  which  they  are 
tested.  In  the  lower  grades  of  virulence  a  local  swelling  only 
results;  in  the  higher,  death  ensues  in  from  twenty- four  to  forty- 
eight  hours;  in  the  case  of  very  high  degrees  of  virulence,  much 


80  PATHOGENIC   BACTERIA 

smaller  doses  of  such  a  broth  culture  are  lethal  within  the  same 
time;  death  may  be  deferred  for  seven  or  even  twenty-one  days 
when  the  virulence  is  low. 

The  crucial  test,  however,  as  to  whether  a  bacillus  is  truly  a 
diphtheritic  one  is  not,  strictly  speaking,  its  possession  in  gen- 
eral of  a  pathogenic  property,  but  of  one  so  specific  that  the  lo- 
cal or  general  action  of  a  broth  culture  is  inhibited  by  the  pre- 
vious injection  of  the  animal  with  anti-diphtheritic  serum. 

The  true  diphtheria  bacilli,  again,  are  characterized  physi- 
ologically by  producing  an  acid  reaction  in  a  1  per  cent,  glucose 
broth.  This  reaction  is  not  given,  e.g.,  by  Hof mann's  bacillus — 
a  form  sometimes  present  along  with  that  of  true  diphtheria,  or 
met  with  in  cases  of  sore  throat  where  none  of  the  true  forms 
occur  (see  Plate). 

Lastly,  it  is  a  fact,  with  much  practical  bearing,  that  bacilli 
having  the  morphological  characters  of  those  of  diphtheria,  and 
highly  virulent,  as  tested  upon  the  guinea  pig,  may  be  isolated 
from  the  throats  of  persons,  who  themselves  exhibit  no  disease 
and  have  not  suffered  from  diphtheria;  since  such  individuals, 
whilst  themselves  immune,  may  serve  as  carriers  of  infection. 
This  has  been  particularly  shown  in  the  case  of  outbreaks  of 
diphtheria  in  schools. 

And,  what  is  equally  important,  after  a  diphtheritic  patient 
has  quite  recovered  from  the  disease,  that  is  to  say,  after  having 
acquired  an  immunity  from  the  disease  by  reason  of  having  had 
it,  he  may  bear  about  diphtheria  bacilli  in  the  throat  for  months, 
harmless  enough  to  himself,  but  capable  of  conveying  the  dis- 
ease to  others.  It  becomes,  hence,  strictly  necessary  to  make 
repeated  bacteriological  examinations  of  the  throat  after  conva- 
lescence is  established;  and  if  the  patient  is  to  be  no  longer  a 


OF  THE   HUMAN   SUBJECT.  81 

source  of  danger  to  others,  his  isolation  may  be  maintained  so 
long  as  the  bacilli  persist. 

Formulae  of  the  stains  referred  to  in  this  volume  : 

Carlol fuchsin  (Neelsen's  solution'): 


Fuchsin  1  gram 

Absolute  Alcohol 


Aqueous  solution  of  Carbolic 


10  cubic  centimeters  (c.c.) 

Aqueous  solution  of  methylene  Hue: 

ylene  Blue  2  £ 

lute  Alcohol  15  c.c. 


Acid  (5  per  cent.)        100  c.c 


Methylene  Blue  2  grams  |  Distilled  Water  85  c.c. 

Absolu 


Loffler's  methylene  blue : 
Concentrated  alcoholic  solu- 


tion of  Methylene  Blue 


30  c.c. 


Solution  of  Caustic  Potash  in 
distilled  water  (1  in  10,000) 
100  c.c. 


Aqueous  solution  of  gentian  violet : 
Gentian  Violet        2.25  gram  |  Distilled  Water  -100  c.c. 

Or  the  following  may  be  used,  except  for  the  coloration  of 
the  hanging  drop  (see  p.  28). 

Gentian  Violet  1  gram  I  Distilled  Water  80  c.c. 

Absolute  Alcohol   20  c.c.          | 

As  gentian  violet  is  a  basic  dye,  the  washing  of  cover-glasses 
after  staining  with  an  aqueous  solution  of  this  re-agent  is  best 
carried  out  in  tap  water,  which  is  naturally  slightly  alkaline; 
distilled  water  extracts  more  of  the  dye,  and  acidulated  water 
yet  more.  Better  than  tap  water,  however,  is  a  solution  of  caus- 
tic potash  in  distilled  water,  one  in  10,000,  the  use  of  which  for 
this  purpose  was  devised  by  Mr.  Arthur  Mead.  The  cover- 
glasses,  after  staining,  are  passed  through  two  saucers  of  the 
potash  solution,  and  finally  through  tap  water. 


82  PATHOGENIC   BACTERIA. 

A  series  of  comparative  trials  has  shown  that  the  violet  is  in 
this  way  rendered  more  intense,  and  the  result  of  the  stain  more 
certain.  Or  the  potash,  as  suggested  by  the  author,  may  be 
added  to  the  dye.  The  following  formula  of  Mr.  Mead's  gives 
excellent  results,  the  specimens  being  washed  in  tap  water  after 
the  use  of  the  stain. 


Potassic  gentian  violet : 
Gentian  Violet    .          1  gram 
Absolute  Alcohol   20  c.c. 


Caustic  Potash  Solution  in  dis- 
tilled water  (1  in  10,000) 


100  c.c. 
Concentrated  alcoholic  solution  of  gentian  violet : 

Gentian  Violet  25  gram  |  Absolute  Alcohol          100  c.c. 

Gram's  method :  * 

(1)  Aniline  gentian  violet : 

Concentrated  alcoholic   solu-  I  Aniline  Water  1,000  c.c. 

tion  of  Gentian  Violet  11  c.c.  | 

The  solution  is  to  be  freshly  made,  and  filtered  before  use. 
Aniline  water  is  prepared  by  well  shaking  4  c.c.  of  pure  aniline 
with  100  c.c.  of  distilled  water,  and  twice  filtering  through 
paper,  first  moistened  with  distilled  water. 

(2)  Iodine  solution : 

Iodine  1  gram  I  Distilled  Water  300  c.c. 

Iodide  of  Potassium  2 

The  preparation  of  distilled  water  for  the  purpose  of 
making  cover-glass  films  (described  on  p.  26),  may  be  ex- 
peditiously  carried  out  by  allowing  the  steam  of  a  beaker  or 
kettle  to  condense  on  the  clean  under  side  of  a  flat  capsule,  par- 
tially filled  with  cold  water,  and  afterward  quickly  inverting  the 
latter,  when  ample  will  be  found  on  the  side  brought  uppermost. 

*  [The  thin  smear  is  dried  on  the  cover-glass,  fixed  by  heat,  stained  with  aniline 
gentian  violet  for  five  minutes,  immersed  in  the  iodine  solution  one  or  two  minutes, 
washed  in  strong  alcohol  until  nearly  colorless,  dried  and  mounted  with  balsam. 


I.U 


